U.S. patent application number 14/369741 was filed with the patent office on 2015-01-08 for crlf-2 binding peptides, protocells and viral-like particles useful in the treatment of cancer, including acute lymphoblastic leukemia (all).
This patent application is currently assigned to STC.UNM. The applicant listed for this patent is SANDIA CORPORATION, STC. UNM. Invention is credited to Carlee Erin Ashley, C. Jeffrey Brinker, Eric C. Carnes, Robert Eric Castillo, Bryce Chackerian, Katherine Epler, David S. Peabody, Walker Kip Wharton, Cheryl L. Willman.
Application Number | 20150010475 14/369741 |
Document ID | / |
Family ID | 48745381 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010475 |
Kind Code |
A1 |
Brinker; C. Jeffrey ; et
al. |
January 8, 2015 |
CRLF-2 BINDING PEPTIDES, PROTOCELLS AND VIRAL-LIKE PARTICLES USEFUL
IN THE TREATMENT OF CANCER, INCLUDING ACUTE LYMPHOBLASTIC LEUKEMIA
(ALL)
Abstract
The present invention relates to the use of which are attached
or anchored phospholipid biolayers further modified by CRLF-2 and
CD 19 binding peptides which may be used for delivering
pharmaceutical cargos, to cells expressing CRLF-2 and CD 19,
thereby treating cancer, in particular, acute lymphoblastic
leukemia (ALL), including (B-precursor acute lymphoblastic leukemia
(B-ALL). Novel CRLF-2 binding peptides and CLRF-2 and CD19-binding
viral-like particles (VLPs) useful in the treatment of cancer,
including ALL are also provided.
Inventors: |
Brinker; C. Jeffrey;
(Albuquerque, NM) ; Peabody; David S.;
(Albuquerque, NM) ; Wharton; Walker Kip;
(Corrales, NM) ; Chackerian; Bryce; (Albuquerque,
NM) ; Ashley; Carlee Erin; (Albuquerque, NM) ;
Willman; Cheryl L.; (Albuquerque, NM) ; Carnes; Eric
C.; (Albuquerque, NM) ; Epler; Katherine;
(Albuquerque, NM) ; Castillo; Robert Eric;
(Albuquerque, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STC. UNM
SANDIA CORPORATION |
Albuquerque
ALBUQUERQUE |
NM
NM |
US
US |
|
|
Assignee: |
; STC.UNM
Albuquerque
MX
SANDA CORPORATION
ALBUQUERQUE
MX
|
Family ID: |
48745381 |
Appl. No.: |
14/369741 |
Filed: |
December 31, 2012 |
PCT Filed: |
December 31, 2012 |
PCT NO: |
PCT/US12/72297 |
371 Date: |
June 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61581915 |
Dec 30, 2011 |
|
|
|
Current U.S.
Class: |
424/9.6 ;
424/450; 514/34; 514/44A; 514/44R; 530/329 |
Current CPC
Class: |
C12N 15/111 20130101;
A61K 31/704 20130101; C12N 15/113 20130101; A61P 35/00 20180101;
A61K 47/62 20170801; A61K 47/64 20170801; A61K 47/6911 20170801;
C07K 7/06 20130101 |
Class at
Publication: |
424/9.6 ;
530/329; 424/450; 514/34; 514/44.R; 514/44.A |
International
Class: |
A61K 47/48 20060101
A61K047/48; C12N 15/113 20060101 C12N015/113; C12N 15/11 20060101
C12N015/11; C07K 7/06 20060101 C07K007/06; A61K 31/704 20060101
A61K031/704 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under the
NIH/Roadmap for Medical Research under grant PHS 2 PN2 EY016570B;
NCI Cancer Nanotechnology Platform Partnership grant
1U01CA151792-01; the Air Force Office of Scientific Research grant
9550-10-1-0054; the U.S. Department of Energy, Office of Basic
Energy Sciences, Division of Materials Sciences and Engineering;
the Sandia National Laboratories' Laboratory Directed Research and
Development (LDRD) program; the President Harry S. Truman
Fellowship in National Security Science and Engineering at Sandia
National Laboratories (C.E.A.). Accordingly, the United States has
certain rights in the invention.
Claims
1. A CRLF-2 binding peptide consisting essentially of a peptide
according to the sequence MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID
NO:5), AAQTSTP (SEQ ID NO:6), TDAHASV (SEQ ID NO:7), FSYLPSH (SEQ
ID NO: 8), YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ ID NO:10), AATLFPL
(SEQ ID NO:11), LTSRPTL (SEQ ID NO:12), ETKAWWL (SEQ ID NO:13)
HWGMWSY (SEQ ID NO:14), SQIFGNK (SEQ ID NO:15), SQAFVLV (SEQ ID
NO:16), WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID NO:18), TMCIYCT
(SEQ ID NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW (SEQ ID NO:21) or
NILSLSM (SEQ ID NO:22).
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. A cell-targeting porous protocell comprising: a nanoporous
silica or metal oxide core with a supported lipid bilayer and at
least one further component selected from the group consisting of a
CRLF-2 binding peptide according to claim 1 which is covalently
linked or complexed to the surface of said protocell; a fusogenic
peptide that promotes endosomal escape of protocells and
encapsulated DNA, and at least one additional cargo component
selected from the group consisting of double stranded linear DNA;
plasmid DNA; an anticancer drug; an imaging agent, small
interfering RNA, small hairpin RNA, microRNA, or a mixture thereof,
wherein one of said cargo components is optionally conjugated
further with a nuclear localization sequence.
7. The protocell according to claim 6 wherein said additional cargo
component is an anti-cancer drug and said lipid bilayer is fused to
said nanoporous core.
8. The protocell according to claim 7 wherein said anticancer drug
is selected from the group consisting of doxorubicin,
5-fluorouracil, cisplatin, cyclophosphamide, vincristin (oncovin),
vinblastine, prednisolone, procarbazine, L-asparaginase,
cytarabine, hydroxyurea, 6-mercaptopurine, methotrexate,
6-thioguanine, bleomycin, etoposide, ifosfamide and mixtures
thereof.
9. (canceled)
10. The protocell according to claim 6 wherein said fusogenic
protein consists essentially of H5WYG peptide (SEQ ID NO: 24) or an
eight mer of polyarginine (SEQ ID NO: 23).
11. (canceled)
12. The protocell according to claim 6 comprising plasmid DNA,
wherein said plasmid DNA is optionally modified to express a
nuclear localization sequence.
13. The protocell according to claim 12 wherein said plasmid DNA is
supercoiled or packaged plasmid DNA
14. (canceled)
15. The protocell according to claim 12 wherein said plasmid DNA is
modified to express a nuclear localization sequence.
16. The protocell according to claim 12 wherein said DNA is
histone-packaged supercoiled plasmid DNA comprises a mixture of
human histone proteins.
17. (canceled)
18. (canceled)
19. The protocell according to claim 6 wherein said plasmid DNA is
capable of expressing a polypeptide toxin, a small hairpin RNA
(shRNA) or a small interfering RNA (siRNA).
20. The protocell according to claim 19 wherein said polypeptide
toxin is selected from the group consisting of ricin toxin A chain,
diphtheria toxin A chain or cholera toxin A chain.
21. (canceled)
22. (canceled)
23. (canceled)
24. The protocell according to claim 6 wherein said nuclear
localization sequence is a peptide according to SEQ ID NO: 28, SEQ
ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
25. (canceled)
26. A CRLF-2 and/or CD-19 targeting protocell comprising: (a) a
core comprising a plurality of negatively-charged, nanoporous,
nanoparticulate silica cores that are optionally modified with an
amine-containing silane and that are interspersed with one or more
anticancer agents that are useful in the treatment of a cancer that
overexpresses CRLF-2 and/or CD-19; and (b) a lipid bilayer which
encapsulates the core and which comprises one of more lipids
selected from the group consisting of
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleylglycero
triethyleneglycyl iminodiacetic acid (DOIDA),
distearylglycerotriethyleneglycyl iminodiacetic acid (DOIDA),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof, wherein the lipid bilayer comprises
a cationic lipid and one or more zwitterionic phospholipids and
contains on its surface at least one peptide that targets CRLF-2
and/or CD19.
27. The protocell of claim 26, wherein said peptide that targets
CRLF-2 is a CRLF-2 binding peptide consisting essentially of a
peptide sequence according to claim 1.
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. A CRLF-2 and/or CD 19-targeting protocell comprising: (a) a
core comprising a plurality of negatively-charged, nanoporous,
nanoparticulate silica cores that are optionally modified with an
amine-containing silane such as
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEPTMS) and that
are interspersed with one or more siRNA that are useful in the
treatment of ALL; and (b) a lipid bilayer which encapsulates the
core and which comprises one of more lipids selected from the group
consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof, wherein the lipid bilayer comprises
a cationic lipid and one or more zwitterionic phospholipids and
contains on its surface at least one peptide that targets CRLF-2
and/or CD 19.
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. A pharmaceutical composition comprising a population of
protocells according to claim 1 and a pharmaceutically-acceptable
carrier, additive or excipient.
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
57. A method of treating a subject suffering from a cancer that
overexpresses CLRF-2 acute lymphoblastic leukemia (ALL), the method
comprising administering to the subject a
pharmaceutically-effective amount of a population of protocells
according to claim 6 and, optionally, an additional anti-cancer
agent.
58. The method according to claim 57 wherein said cancer is acute
lymphoblastic leukemia.
59. (canceled)
60. (canceled)
61. A protocell nanostructure comprising: (a) a porous nanoparticle
comprising a plurality of pores; (b) at least one lipid bilayer
surrounding the porous particle to form a protocell; (c) at least
one CRLF-2 targeting peptide according to claim 1 conjugated to
said lipid bilayer; and (d) a cargo component which comprises at
least one therapeutic agent loaded into the protocell nanostructure
for delivery to a patient.
62. The protocell of claim 61, wherein said cargo component
includes at least one component is selected form the group
consisting of small molecules, ShRNA, siRNa or a polypeptide
toxin.
63. (canceled)
64. (canceled)
65. The protocell according to claim 61, wherein said therapeutic
agent is an anticancer agent selected from the group consisting of
everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101,
pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886),
AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib,
ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3
inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora
kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC
inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an
EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a
PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a
checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a
Map kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed,
erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin,
oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab,
zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene,
oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111,
131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan,
IL13-PE38QQR, INO 1001, IPdR.sub.1 KRX-0402, lucanthone, LY 317615,
neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr
311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat,
etoposide, gemcitabine, doxorubicin, liposomal doxorubicin,
5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,
seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,
N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled
irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane,
letrozole, DES (diethylstilbestrol), estradiol, estrogen,
conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258,);
3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone,
vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6,
Azgly 10] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu
t)-Leu-Arg-Pro-Azgly-NH.sub.2 acetate
[C.sub.59H.sub.84N.sub.18Oi.sub.4-(C.sub.2H.sub.4O.sub.2).sub.X
where x=1 to 2.4], goserelin acetate, leuprolide acetate,
triptorelin pamoate, medroxyprogesterone acetate,
hydroxyprogesterone caproate, megestrol acetate, raloxifene,
bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;
TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF
antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide
hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,
sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide,
L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin,
buserelin, busulfan, carboplatin, carmustine, chlorambucil,
cisplatin, cladribine, clodronate, cyproterone, cytarabine,
dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,
epirubicin, fludarabine, fludrocortisone, fluoxymesterone,
flutamide, gemcitabine, hydroxyurea, idarubicin, ifosfamide,
imatinib, leuprolide, levamisole, lomustine, mechlorethamine,
melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin,
mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin,
pamidronate, pentostatin, plicamycin, porfimer, procarbazine,
raltitrexed, rituximab, streptozocin, teniposide, testosterone,
thalidomide, thioguanine, thiotepa, tretinoin, vindesine,
13-cis-retinoic acid, phenylalanine mustard, uracil mustard,
estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine
arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol,
valrubicin, mithramycin, vinblastine, vinorelbine, topotecan,
razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine,
endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862,
angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone,
finasteride, cimitidine, trastuzumab, denileukin diftitox,
gefitinib, bortezimib, paclitaxel, cremophor-free paclitaxel,
docetaxel, epithilone B, BMS-247550, BMS-310705, droloxifene,
4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,
fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424,
HMR-3339, ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352,
rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573,
RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684,
LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim,
darbepoetin, erythropoietin, granulocyte colony-stimulating factor,
zolendronate, prednisone, cetuximab, granulocyte macrophage
colony-stimulating factor, histrelin, pegylated interferon alfa-2a,
interferon alfa-2a, pegylated interferon alfa-2b, interferon
alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,
hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,
all-transretinoic acid, ketoconazole, interleukin-2, megestrol,
immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab
tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene,
tositumomab, arsenic trioxide, cortisone, editronate, mitotane,
cyclosporine, liposomal daunorubicin, Edwina-asparaginase,
strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,
palonosetron, aprepitant, diphenhydramine, hydroxyzine,
metoclopramide, lorazepam, alprazolam, haloperidol, droperidol,
dronabinol, dexamethasone, methylprednisolone, prochlorperazine,
granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim,
erythropoietin, epoetin alfa, darbepoetin alfa and mixtures
thereof
66. (canceled)
67. (canceled)
68. (canceled)
69. (canceled)
70. (canceled)
71. The protocell according to claim 61, wherein said targeting
peptide is any of the CRLF-2 targeting peptides as set forth in
attached FIGS. 10-14 hereof.
72. The protocell according to claim 61, wherein said targeting
peptide is a consensus sequence according to SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:34 or SEQ ID NO:35.
73. A CRLF-2 binding peptide sequence as set forth in any of FIGS.
10-14 hereof.
74. (canceled)
75. (canceled)
76. (canceled)
77. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Patent Application Ser. No. 61/581,915, filed Dec. 30,
2011, and entitled "CRLF-2 Binding Peptides and CRLF-2-Targeted
VLPs for Leukemia Therapy", the complete contents of which
provisional application is incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to the use of which are
attached or anchored phospholipid biolayers further modified by
CRLF-2 and CD 19 binding peptides which may be used for delivering
pharmaceutical cargos, to cells expressing CRLF-2 and CD19, thereby
treating acute lymphoblastic leukemia (B-precursor acute
lymphoblastic leukemia (ALL)). Novel CRLF-2 and CD19-binding
viral-like particles (VLPs) useful in the treatment of ALL are also
provided.
[0004] The present invention also relates to the specific
oligopeptides which bind CRLF-2 CD 19 and can be used in numerous
applications (therapeutic, diagnostic and the like) to treat
disease states and/or conditions which are modulated through or
occur in a cell which expresses CRLF-2 or CD 19. In the present
invention, protocells which comprise on their surface a
phospholipid bilayer and at least one CRLF-2 or CD19 binding
peptide which binds to CRLF-2 or CD19 on a cell to which the
protocell binds, and through endocytosis or other mechanism, the
contents of the protocell is released into the targeted cell
resulting in apoptosis or other cellular degradation and/or
inhibition to effect an intended therapeutic result.
BACKGROUND OF THE INVENTION
[0005] Acute lymphoblastic leukemia ("ALL", also referred to as
"childhood leukemia, of which B-precursor acute lymphoblastic
leukemia (B-ALL) is the most common form is a disease characterized
by the uncontrolled proliferation of malignant lymphocytes leading
to the suppression of normal hematopoiesis, is the most frequently
diagnosed cancer in children. Current therapies result in the
induction of long term remission in 80% of pediatric ALL patients.
However, death from relapsed ALL remains the second leading cause
of mortality in children (surpassed only by deaths caused by
accidents). In addition, children who enter remission suffer from
significant life altering short- and long-term complications due to
the side effects of the cytotoxic therapies. Therefore new
generations of therapies are required both to enhance survival and
improve quality of life in pediatric ALL patients. R. Harvey, C.
Willman et el., Blood 2010 have shown that preferential expression
of CRLF2 surface markers is associated specific cohorts of
pediatric ALL with "poor outcome".
[0006] The delivery of cancer therapeutic agents sequestered in
nanoparticles has the potential to bypass many severe problems
associated with systemic drug administration. .sup.1,2
Encapsulation allows treatment with compounds that are poorly
soluble and/or unstable in physiological solutions, as well as
those that are rapidly metabolized or cleared when administered as
free drugs. Conjugation of the particle with a targeting moiety
that recognizes an antigen over-expressed on the surface of a tumor
cell results in a series of additional benefits, including the
limitation of damage to normal cells and a marked dose escalation
that results from the localized release of highly concentrated
drugs at the site of a tumor or within a cancer cell.
[0007] However, the therapeutic potential of many classes of
macromolecules, especially nucleic acids and proteins, is severely
limited because of degradation by plasma enzymes or an induction of
an immune response following systemic administration. In addition,
cellular uptake is typically restricted due to issues with either
size or charge. The ability to package these molecules within
particles overcomes such impediments and allows evaluation of the
therapeutic efficacy of a large number of agents not presently
available for clinical applications.
[0008] For example, the therapeutic potential of numerous
anti-cancer and other therapeutic agents, including small and
macromolecules, including traditional small molecule anticancer
agents, as well as macromolecular compounds such as small
interfering RNAs (siRNAs, which interfere with/silence expression
of various cyclins in the cell (e.g., one or more of cyclin A2,
cyclin B1, cyclin D1 or cyclin E1, among others) and protein toxins
is severely limited by the availability of delivery platforms that
prevent degradation and non-specific interactions during
circulation but promote uptake and intracellular trafficking in
targeted cells.
[0009] Despite tremendous advances, two primary challenges remain
for the successful treatment of pediatric ALL. With the intensity
of therapy now tailored to a child's relapse risk, nearly 80% of
children survive. Yet to achieve these levels of cure, children are
exposed to very intensive systemic chemotherapeutic regimens which
are frequently associated with significant toxicities and serious
short and long-term side effects. Thus, more targeted, less toxic
treatments for ALL are needed. Secondly, 25% of children still
relapse despite receiving intensive therapy and ALL remains the
leading cause of cancer death in children; this is particularly
true for the 30% of patients with high-risk forms of disease. More
effective treatments for high-risk ALL are therefore required.
SUMMARY OF THE INVENTION
[0010] The inventors have identified subtypes of ALL patients who
have extremely poor outcomes (<25% EFS). One such group is
characterized by genomic rearrangements of CRLF2 leading to
markedly elevated (upregulated) levels of CRLF2 (the TSLP receptor)
expression on leukemic blasts, making CRLF2 an attractive
therapeutic target in high-risk ALL. CRLF2 rearrangements are
frequently associated with activating mutations in the JAK kinase,
deletion of IKZF1/IKAROS and other genes, Hispanic/American Indian
race and ethnicity, and a very poor outcome.6-10 While virtually
all ALL cases with CRLF2 genomic rearrangements have an "activated
tyrosine kinase" gene expression profile, only half have JAK family
mutations. Our ongoing transcriptomic sequencing studies in the NCI
TARGET Project have revealed that the remaining CRLF2-expressing
cases have other activating novel translocations or genomic
rearrangements (PDGFR, EPOR, JAK, and ABL).
[0011] The inventors recently described a novel and remarkably
versatile nanoparticle, termed a protocell (see FIG. 1), which
synergistically combines features of both mesoporous silica
particles and liposomes to exhibit many features of an ideal
targeted therapeutic delivery platform.
[0012] The protocells are formed via fusion of liposomes to porous
silica nanoparticles. The high pore volume and surface area of the
spherical nanoporous silica core allow high-capacity encapsulation
of a spectrum of cargos. The surrounding lipid bilayer, whose
composition can be modified for specific biological applications,
serves as a modular, reconfigurable scaffold, allowing the
attachment of a variety of molecules that provide cell-specific
targeting and controlled intracellular trafficking. Generally, our
protocells target CRLF-2 and/or CD19 and have a 30- to 100-fold
greater capacity for anticancer agents including siRNA than
corresponding liposomes and are markedly more stable when incubated
under physiological conditions. In certain applications, these
protocells are loaded with low molecular weight therapeutic agents
and conjugated with a peptide that specifically recognizes
hepatocarcinomas induce cytotoxicity with a 10.sup.6-fold
improvement in efficacy compared to corresponding liposomes.
[0013] Embodiments of the present invention are directed to
protocells for specific targeting of cells, in particular aspects,
cancer cells which express high levels of CRLF-2 and/or CD19,
especially cancer cells of acute lymphoblastic leukemia, including
B-cell ALL.
[0014] In certain aspects, the present invention is directed to a
cell-targeting porous protocell comprising a nanoporous silica or
metal oxide core with a supported lipid bilayer, and at least one
further component selected from the group consisting of [0015] a
cell targeting species consisting essentially of a CRLF-2 binding
peptide as otherwise described herein; [0016] a fusogenic peptide
that promotes endosomal escape of protocells and encapsulated DNA,
[0017] other cargo comprising at least one cargo component selected
from the group consisting of double stranded linear DNA or a
plasmid DNA; [0018] a drug; [0019] an imaging agent, [0020] small
interfering RNA, small hairpin RNA, microRNA, or a mixture thereof,
[0021] wherein one of said cargo components is optionally
conjugated further with a nuclear localization sequence.
[0022] In certain embodiments, protocells according to embodiments
of the invention comprise a nanoporous silica core with a supported
lipid bilayer; a cargo comprising at least one therapeutic agent
which optionally facilitates cancer cell death such as a
traditional small molecule (preferably an anticancer agent which is
useful in the treatment of ALL, in particular, B-ALL), a
macromolecular cargo (e.g. siRNA such as S565, S7824 and/or s10234,
among others, shRNA or other micro RNA or a protein toxin such as a
ricin toxin A-chain or diphtheria toxin A-chain) and/or a packaged
plasmid DNA (in certain embodiments--histone packaged) disposed
within the nanoporous silica core (preferably supercoiled as
otherwise described herein in order to more efficiently package the
DNA into protocells as a cargo element) which is optionally
modified with a nuclear localization sequence to assist in
localizing/presenting the plasmid within the nucleus of the cancer
cell and the ability to express peptides involved in therapy (e.g.,
apoptosis/cell death of the cancer cell) or as a reporter
(fluorescent green protein, fluorescent red protein, among others,
as otherwise described herein) for diagnostic applications.
Protocells according to the present invention include a targeting
peptide which targets cells for therapy (e.g., cancer cells in
tissue to be treated) such that binding of the protocell to the
targeted cells is specific and enhanced and a fusogenic peptide
that promotes endosomal escape of protocells and encapsulated DNA.
Protocells according to the present invention may be used in
therapy or diagnostics, more specifically to treat cancer and other
diseases, including viral infections, especially including
childhood acute lymphoblastic leukemia, especially include B-ALL.
In other aspects of the invention, protocells use novel binding
peptides (CRLF-2 binding peptides as otherwise described herein)
which selectively bind to cancer tissue (including leukemia cells,
liver, kidney, bone and non-small cell lung cancer cells,) for
therapy and/or diagnosis of cancer, including the monitoring of
cancer treatment and drug discovery.
[0023] In one aspect, protocells according to embodiments of the
present invention comprise a porous nanoparticle protocell which
often comprises a nanoporous silica core with a supported lipid
bilayer. In this aspect of the invention, the protocell comprises a
targeting peptide which is CRLF-2 receptor binding peptide as
otherwise described herein, often in combination with a fusogenic
peptide on the surface of the protocell. The protocell may be
loaded with various therapeutic and/or diagnostic cargo, including
for example, small molecules (therapeutic and/or diagnostic,
especially including anticancer and/or antiviral agents (for
treatment of HBV and/or HCV), macromolecules including polypeptides
and nucleotides, including RNA (shRNA, siRNA and other micro RNA)
or plasmid DNA which may be supercoiled and histone-packaged
including a nuclear localization sequence, which may be therapeutic
and/or diagnostic (including a reporter molecule such as a
fluorescent peptide, including fluorescent green protein/FGP,
fluorescent red protein/FRP, among others).
[0024] Additional embodiments of the present invention are directed
to Virus-like particles (VLPs) as otherwise described herein which
express CRLF-2 binding peptides as heterologous peptides on the
surface of the VLP, such as that VLP may be used to target cancer
cells and deliver therapeutic cargo in the treatment of cancer, in
particular ALL, including B-ALL.
[0025] Other aspects of embodiments of the present invention are
directed to pharmaceutical compositions. Pharmaceutical
compositions according to the present invention comprise a
population of protocells which may be the same or different and are
formulated in combination with a pharmaceutically acceptable
carrier, additive or excipient. The protocells may be formulated
alone or in combination with another bioactive agent (such as an
additional anti-cancer agent or an antiviral agent) depending upon
the disease treated and the route of administration (as otherwise
described herein). These compositions comprise protocells as
modified for a particular purpose (e.g. therapy, including cancer
therapy, or diagnostics, including the monitoring of cancer
therapy). Pharmaceutical compositions comprise an effective
population of protocells for a particular purpose and route of
administration in combination with a pharmaceutically acceptable
carrier, additive or excipient.
[0026] One aspect of the present invention is directed to the
finding that protocells exhibit multiple properties that overcome
many of the aforementioned limitations in effectively delivering
active ingredients to treat pediatric ALL by targeting CRLF-2
and/or CD 19. Specifically, in certain embodiments of the instant
invention, protocells loaded with a cocktail of anticancer agents
bind to cells in a manner dependent on the presence of an
appropriate targeting peptide for CRLF-2 and/or CD 19, which are
expressed on leukemia cells as well as on other cancer cells and,
through an endocytic pathway, promote delivery of the traditional
chemotherapeutic agents, anticancer agents including siRNAs and
protein toxins silencing nucleotides to the cytoplasm.
[0027] The discovery of novel ALL subtypes, together with our
preliminary studies demonstrating a lack of efficacy of JAK kinase
inhibitors as single agents in our xenograft models of human ALL
containing CRLF2 and JAK mutations, and the observation that a
large percentage of high risk B-precursor ALL samples express
measurable levels of CRLF2 mRNA compared to normal B cells and
respond to TSLP, leads us to hypothesize that CRLF2 is a superior
target for therapy in high-risk ALL.
[0028] In order to expand the universe of potential molecular
targets with a parallel increase in leukemic subtypes that are
amenable to treatment, as well as to allow for simultaneous
targeting with multiple classes of particles, we also describe
novel protocells engineered to target molecules expressed on a
wider class of ALL blasts and B cell malignancies, including CD19
and CD22.
[0029] In one embodiment, the invention provides a porous
nanoparticle protocell which comprises a nanoporous silica core
with a supported lipid bilayer and a peptide as described herein
which targets CRLF-2 and/or CD 19. Preferably, the protocell
surface comprises a fusogenic peptide. The protocell may be loaded
with various therapeutic and/or diagnostic cargo, including for
example, small molecules that are useful in the treatment of
pediatric ALL, macromolecules including polypeptides and
nucleotides, including RNA (shRNA, siRNA or other micro RNA) or
plasmid DNA which may be supercoiled and histone-packaged including
a nuclear localization sequence, which may be therapeutic and/or
diagnostic (including a reporter molecule such as a fluorescent
peptide, including fluorescent green protein/FGP, fluorescent red
protein/FRP, among others).
[0030] The nanoporous silica-particle core of the protocells has a
high surface area, a readily variable porosity, and a surface
chemistry that is easily modified. These properties make the
protocell-core amenable to high-capacity loading of many different
types of cargo. The protocell's supported lipid bilayer (SLB) has
an inherently low immunogenicity. Additionally, the SLB provides a
fluid surface to which peptides, polymers and other molecules can
be conjugated in order to facilitate targeted cellular uptake.
These biophysical and biochemical properties allow for the
protocell to be optimized for a specific environment and enable
delivery of disparate types of cargo by a wide variety of
routes.
[0031] In one embodiment, the invention provides a CRLF-2 and/or CD
19-targeting protocell comprising:
(a) a core comprising a plurality of negatively-charged,
nanoporous, nanoparticulate silica cores that are optionally
modified with an amine-containing silane such as
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEPTMS) and that
are interspersed with one or more anticancer agents that are useful
in the treatment of ALL; and (b) a lipid bilayer which encapsulates
the core and which comprises one of more lipids selected from the
group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
dioleylglycero triethyleneglycyl iminodiacetic acid (DOIDA),
distearylglycerotriethyleneglycyl iminodiacetic acid (DSIDA),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof, wherein the lipid bilayer comprises
a cationic lipid and one or more zwitterionic phospholipids and
contains on its surface at least one peptide as otherwise described
herein that targets CRLF-2 and/or CD19 (e.g.
H.sub.2N-MTAAPVHGGHHHHHH-COOH SEQ ID NO:1 or numerous 7 mer
peptides including MTAAPVH SEQ ID NO:4 as otherwise described
herein).
[0032] In certain embodiments, the lipid bilayer's surface also
contains an R8 peptide (e.g. RRRRRRRR SEQ ID NO:23 or as modified
for crosslinking/conjugation with protocells according to the
present invention H.sub.2N-RRRRRRRRGGC-COOH SEQ ID NO:2 or
equivalents thereof) and/or an endosomolytic peptide (H5WYG) (e.g.
GLFHAIAHFIHGGWHGLIHGWY SEQ ID NO:24 or as modified for
crosslinking/conjugation with protocells according to the present
invention H.sub.2N-GLFHAIAHFIHGGWHGLIHGWYGGGC-COOH SEQ ID NO:3 or
equivalents thereof).
[0033] In certain embodiments, the one or more anticancer agents
that are useful in the treatment of ALL are preferably selected
from the group consisting of doxorubicin, 5-fluorouracil,
cisplatin, cyclophosphamide, vincristin (oncovin), vinblastine,
prednisolone, procarbazine, L-asparaginase, cytarabine,
hydroxyurea, 6-mercaptopurine, methotrexate, 6-thioguanine,
bleomycin, etoposide, ifosfamide, sirolomus, quercetin and mixtures
thereof. Preferably, one or more of doxorubicin, 5-fluoruracil
and/or cisplatin are used as anticancer agents in the present
invention for the treatment of ALL.
[0034] In the embodiment of the preceding paragraph, the lipid is
preferably selected from the group consisting of
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) or
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and mixtures
thereof, and the protocell has at least one of the following
characteristics: a BET surface area of greater than about 600
m.sup.2/g, a pore volume fraction of between about 60% to about
70%, a multimodal pore morphology composed of pores having an
average diameter of between about 20 nm to about 30 nm,
surface-accessible pores interconnected by pores having an average
diameter of between about 5 nm to about 15 nm. Preferably, the
protocell encapsulates siRNA wherein the protocell targets CRLF-2
and/or CD19 in an amount of about 0.1 nM to about 10 .mu.M or more,
about 1 to about 500 nM, about 5 to about 100 nM, about 5 to about
25 nM, about 10 nM of siRNA per 10.sup.10 nanoparticulate silica
cores.
[0035] In still another embodiment, the invention provides a CRLF-2
and/or CD 19-targeting protocell comprising:
(a) a core comprising a plurality of negatively-charged,
nanoporous, nanoparticulate silica cores that are optionally
modified with an amine-containing silane such as
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEPTMS) and that
are interspersed with one or more anticancer agents that are useful
in the treatment of ALL; and (b) a lipid bilayer which encapsulates
the core and which comprises one of more lipids selected from the
group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof, wherein (1) the lipid bilayer
comprises a cationic lipid and one or more zwitterionic
phospholipids and contains on its surface at least one peptide that
targets CRLF-2 and/or CD19 ("CRLF-2 binding peptide" e.g. MTAAPVH
SEQ ID NO: 4 or as modified for complexation,
H.sub.2N-MTAAPVHGGHHHHHH-COOH SEQ ID NO:1 or equivalents thereof as
otherwise described herein) (2) the lipid bilayer is loaded with
SP94 and an endosomolytic peptide, and (3) the protocell
selectively binds to a hepatocellular carcinoma cell by targeting
CRLF-2 and/or CD 19.
[0036] In a preferred embodiment of the preceding paragraph, the
lipid bilayer preferably comprises DOPC/DOPE/cholesterol/PEG-2000
in an approximately 55:5:30:10 mass ratio.
[0037] In still another embodiment, the invention provides a CRLF-2
and/or CD 19-targeting protocell comprising:
(a) a core comprising a plurality of negatively-charged,
nanoporous, nanoparticulate silica cores that are optionally
modified with an amine-containing silane such as
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEPTMS) and that
are interspersed with one or more small hairpin RNA (shRNA) and/or
small interfering RNA (siRNA), other micro RNA that are useful in
the treatment of ALL; and (b) a lipid bilayer which encapsulates
the core and which comprises one of more lipids selected from the
group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof, wherein the lipid bilayer comprises
a cationic lipid and one or more zwitterionic phospholipids and
contains on its surface at least one peptide that targets CRLF-2
and/or CD19 (e.g. MTAAPVH SEQ ID NO:4 or as modified, for
complexing with protocells H.sub.2N-MTAAPVHGGHHHHHH-COOH SEQ ID
NO:1 or other 7mer peptides or equivalents as described and/or
modified).
[0038] In certain embodiments of the protocells of the invention,
the lipid bilayer comprises
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) a polyethylene
glycol (PEG), a targeting peptide, and R8 (SEQ ID NO:23), and the
mesoporous, nanoparticulate silica cores each have an average
diameter of around 100 nm, an average surface area of greater than
1,000 m.sup.2/g and surface-accessible pores having an average
diameter of between about 20 nm to about 25 nm, and have a siRNA
load of around 1 .mu.M per 10.sup.10 particles or greater.
[0039] The targeting peptide preferably is a peptide that binds to
CRLF-2 and/or CD 19 as set forth in any of FIGS. 10-14 hereof, and
most preferably consists essentially of a 7-mer peptide sequence
selected from the group consisting of MTAAPVH (SEQ ID NO: 4),
LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID NO:6), TDAHASV (SEQ ID
NO:7), FSYLPSH (SEQ ID NO: 8), YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ
ID NO:10), AATLFPL (SEQ ID NO:11), LTSRPTL (SEQ ID NO:12), ETKAWWL
(SEQ ID NO:13), HWGMWSY (SEQ ID NO:14), SQIFGNK (SEQ ID NO:15),
SQAFVLV (SEQ ID NO:16), WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID
NO:18), TMCIYCT (SEQ ID NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW
(SEQ ID NO:21) and NILSLSM (SEQ ID NO:22). Preferred CRLF-2 binding
peptides include MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5),
AAQTSTP (SEQ ID NO:6), MHAPPFY (SEQ ID NO:10), ETKAWWL (SEQ ID
NO:13), SQIFGNK (SEQ ID NO:15), AATLFPL (SEQ ID NO:11), TDAHASV
(SEQ ID NO:7) and FSYLPSH (SEQ ID NO: 8) or equivalents thereof.
More preferably, the CRLF-2 binding peptide is MTAAPVH (SEQ ID NO:
4), LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID NO:6) or MHAPPFY (SEQ ID
NO:10). Often, the CRLF-2 binding peptide used in embodiments
according to the present invention includes MTAAPVH (SEQ ID NO: 4)
and LTTPNWV (SEQ ID NO:5). Most often, the CRLF-2 binding peptide
is MTAAPVH (SEQ ID NO: 4) or equivalents thereof. Most preferably,
the protocell comprises around 0.01 to around 0.02 wt % of MTAAPVH
(SEQ ID NO: 4), around 10 wt % PEG-2000 and around 0.500 wt % of
R8, SEQ ID. NO: 23.
[0040] In still another aspect, the invention relates to novel
viral-like particles (VLPs) that target CRLF-2 and/or CD19.
Preferably, the VLPs are comprised of a coat polypeptide of the
bacteriophages PP7 or MS2, wherein the coat protein is modified by
insertion of heterologous peptides that target CRLF-2 and/or CD 19,
and wherein the peptides that target CRLF-2 and/or CD19 are
displayed on the VLP and encapsidate PP7 or MS2 mRNA. These
peptides include MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5),
AAQTSTP (SEQ ID NO:6), TDAHASV (SEQ ID NO:7), FSYLPSH (SEQ ID NO:
8), YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ ID NO:10), AATLFPL (SEQ ID
NO:11), LTSRPTL (SEQ ID NO:12), ETKAWWL (SEQ ID NO:13), HWGMWSY
(SEQ ID NO:14), SQIFGNK (SEQ ID NO:15), SQAFVLV (SEQ ID NO:16),
WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID NO:18), TMCIYCT (SEQ ID
NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW (SEQ ID NO:21) and NILSLSM
(SEQ ID NO:22). Preferred CRLF-2 binding peptides include MTAAPVH
(SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID NO:6),
MHAPPFY (SEQ ID NO:10), ETKAWWL (SEQ ID NO:13), SQIFGNK (SEQ ID
NO:15), AATLFPL (SEQ ID NO:11), TDAHASV (SEQ ID NO:7) and FSYLPSH
(SEQ ID NO: 8) or equivalents thereof. More preferably, the CRLF-2
binding peptide is MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5),
AAQTSTP (SEQ ID NO:6) or MHAPPFY (SEQ ID NO:10). Often, the CRLF-2
binding peptide used in embodiments according to the present
invention includes MTAAPVH (SEQ ID NO: 4) and LTTPNWV (SEQ ID
NO:5). Most often, the CRLF-2 binding peptide is MTAAPVH (SEQ ID
NO: 4) or equivalents thereof
[0041] In still another aspect, the invention relates to a
population of viral-like particles (VLPs), each of the viral-like
particles comprising a bacteriophage dimer coat polypeptide on
which is displayed (in the A-B loop, or at the carboxy or amino
terminus of the coat polypeptide) one or more CRLF-2 targeting
peptides or alternatively, single chain, variable fragments of
antibodies that target a CRLF-2 and/or CD 19 epitope, wherein the
one or more viral-like particles each encapsidate (1) mRNA encoding
the bacteriophage, and (2) one or more anticancer agents that are
useful in the treatment of ALL, preferably selected from the group
consisting of doxorubicin, 5-fluorouracil, cisplatin,
cyclophosphamide, vincristin (oncovin), vinblastine, prednisolone,
procarbazine, L-asparaginase, cytarabine, hydroxyurea,
6-mercaptopurine, methotrexate, 6-thioguanine, bleomycin,
etoposide, ifosfamide and mixtures thereof. Preferably, one or more
of doxorubicin, 5-fluoruracil and/or cisplatin is used as the
anticancer agent for treatment of ALL. The bacteriophage is
preferably selected from the group consisting of MS2, Qb, R17, SP,
PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNA
bacteriophages. Preferably, the bacteriophage is a MS2 or PP7
bacteriophage.
[0042] Pharmaceutical compositions according to the present
invention comprise a population of protocells or VLPs which may be
the same or different and are formulated in combination with a
pharmaceutically acceptable carrier, additive or excipient. The
protocells may be formulated alone or in combination with another
bioactive agent (such as an additional anti-cancer agent) depending
upon the route of administration (as otherwise described herein).
These compositions comprise protocells or VLPs as modified for a
particular purpose (e.g. therapy, including cancer therapy, or
diagnostics, including the monitoring of cancer therapy).
Pharmaceutical compositions comprise an effective population of
protocells or VLPs for a particular purpose and route of
administration in combination with a pharmaceutically acceptable
carrier, additive or excipient.
[0043] In further alternative aspects, the present invention
relates to methods of diagnosing cancer, including pediatric ALL,
the method comprising administering a pharmaceutical composition
comprising a population of protocells or VLPs which have been
modified to deliver a diagnostic agent or reporter imaging agent
selectively to cancer cells to identify cancer, including pediatric
ALL in the patient. In this method, protocells or VLPs according to
the present invention may be adapted to target cancer cells,
including pediatric ALL cancer cells through the inclusion of at
least one targeting peptide which binds to CRLF-2 and/or CD 19 and
through the inclusion of a reporter component (including an imaging
agent) of the protocell targeted to the cancer cell, may be used to
identify the existence and size of cancerous tissue in a patient or
subject by comparing a signal from the reporter with a standard.
The standard may be obtained for example, from a population of
healthy patients or patients known to have cancer, including
pediatric ALL. Once diagnosed, appropriate therapy with
pharmaceutical compositions according to the present invention, or
alternative therapy may be implemented.
[0044] In still other aspects of the invention, the compositions
according to the present invention may be used to monitor the
progress of therapy of cancer, including pediatric ALL, including
therapy with compositions according to the present invention. In
this aspect of the invention, a composition comprising a population
of protocells which are specific for cancer, including pediatric
ALL cancer cell binding and include a reporter component may be
administered to a patient or subject undergoing therapy such that
progression of the therapy of cancer, including pediatric ALL can
be monitored.
[0045] Alternative aspects of the invention relate to novel CRLF-2
and/or CD19 binding peptides as otherwise described herein, which
can be used as targeting peptides on protocells of certain
embodiments of the present invention, or in pharmaceutical
compositions for their benefit in binding CRLF-2 and/or CD 19
protein in cancer cells, including hepatocellular cancer cells, and
including pediatric ALL cancerous tissue. One embodiment of the
invention relates to different mer peptides (preferably, 7 mer
peptides) which show activity as novel binding peptides for CRLF-2
and/or CD19 receptors. These peptides are summarized in FIG. 3 and
FIGS. 10-14 and include MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID
NO:5), AAQTSTP (SEQ ID NO:6), TDAHASV (SEQ ID NO:7), FSYLPSH (SEQ
ID NO: 8), YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ ID NO:10), AATLFPL
(SEQ ID NO:11), LTSRPTL (SEQ ID NO:12), ETKAWWL (SEQ ID NO:13),
HWGMWSY (SEQ ID NO:14), SQIFGNK (SEQ ID NO:15), SQAFVLV (SEQ ID
NO:16), WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID NO:18), TMCIYCT
(SEQ ID NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW (SEQ ID NO:21) and
NILSLSM (SEQ ID NO:22). Preferred CRLF-2 binding peptides include
MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID
NO:6), MHAPPFY (SEQ ID NO:10), ETKAWWL (SEQ ID NO:13), SQIFGNK (SEQ
ID NO:15), AATLFPL (SEQ ID NO:11), TDAHASV (SEQ ID NO:7) and
FSYLPSH (SEQ ID NO: 8). More preferably, the CRLF-2 binding peptide
is MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID
NO:6) or MHAPPFY (SEQ ID NO:10). Often, the CRLF-2 binding peptide
used in embodiments according to the present invention includes
MTAAPVH (SEQ ID NO: 4) and LTTPNWV (SEQ ID NO:5). Most often, the
CRLF-2 binding peptide is MTAAPVH (SEQ ID NO: 4).
[0046] Each of these peptides may be used alone or in combination
with other CRLF-2 and/or CD 19 binding peptides within the above
group or with a spectrum of other targeting peptides (e.g., SP94
peptides as described herein) which may assist in binding
protocells or VLPs according to an embodiment of the present
invention to pediatric ALL cancer cells, including hepatocellular
cancer cells, ovarian cancer cells, breast cancer cells and
cervical cancer cells, amongst numerous other cancer cells. These
peptides may be formulated alone or in combination with other
bioactive agents for purposes of providing an intended result.
Pharmaceutical compositions can comprise an effective amount of at
least one of the CRLF-2 and/or CD 19-binding peptides identified
above, in combination with a pharmaceutically acceptable carrier,
additive or excipient optionally in combination with an additional
bioactive agent, which may include an anticancer agent or other
bioactive agent.
[0047] Methods of treating subjects suffering from cancer,
including pediatric ALL are also described.
[0048] These and other aspects of the invention are described
further in the Detailed Description of the Invention. In addition,
certain aspects of the present invention have been discussed in
detail in U.S. patent application Ser. No. 11/895,198 (US
Publication 2009/0054246), entitled "A Virus-Like Platform for
Rapid Vaccine Discovery", international patent application
PCT/US2012/035529 (Publication WO2012/149376), entitled "Porous
Nanoparticle-Supported Lipid Bilayers (Protocells) for Targeted
Delivery and Methods of Using Same" and U.S. patent application
Ser. No. 12/960,168, filed Dec. 3, 2010, entitled "Virus-Like
Particles as Targeted Multifunctional Nanocarriers for Delivery of
Drugs, Therapeutics, Sensors and Contrast Agents to Arbitrary Cell
Types", each of which applications is incorporated by reference in
its entirety herein.
BRIEF DESCRIPTION OF THE FIGURES
[0049] FIG. 1 illustrates how a protocell is a flexible platform
for targeted delivery), as determined in the experiment(s) of
Example 1. The TEM image shows that a porous nanoparticle can serve
as a support for lipid bilayers, which in turn seal contents within
the core.
[0050] FIG. 2 illustrates the identification of targeting peptides
in accordance with the invention), as determined in the
experiment(s) of Example 1.
[0051] FIG. 3 shows how flow cytometry is used to evaluate binding
of individual phage clones within consensus sequences. In this
case, a saturatable binding curve can be constructed, allowing for
the determination of the disassociation constant (K.sub.d) of phage
displaying potential specific peptides that bind cells with high
levels of CRLF2 expression but not parental cell lines with minimal
expression), as determined in the experiment(s) of Example 1.
[0052] FIG. 4 illustrates how protocells bind to target cells with
high specificity at low peptide densities due to a fluid supported
bilayer), as determined in the experiment(s) of Example 1.
[0053] FIG. 5 illustrates that once a targeting peptide has been
selected, human cells known to over-express CRLF-2 (MMH CALL 4) are
used to evaluate the binding constants of peptide that has been
cross-linked to protocell-supported lipid bilayer (SLB)), as
determined in the experiment(s) of Example 1. The same targeting
peptide can also be displayed on protocell SLBs featuring mixtures
of lipids which form segregated domains which serve to increase the
local concentration of peptide.
[0054] FIG. 6 shows that targeted protocells can become
internalized within target cells (MMH CALL 4, Mutz-5 and
BaF3/CRLF-2) over time), as determined in the experiment(s) of
Example 1. FIG. 6 also shows that targeted peptides displayed on a
fluid lipid (DOIDA) domain within a non-fluid SLB (DSPC) show high
binding affinity to target cells which over-express CRF-2 (MHH Cell
4, Mutz and BaF3/CRLF-2).
[0055] FIG. 7 shows that targeted protocells can become
internalized within target cells (MHH CALL4, Mutz-5 and
BaF3/CRLF-2) over time), as determined in the experiment(s) of
Example 1.
[0056] FIG. 8 illustrates that targeted protocells that display the
R8 peptide show increased internalization kinetics), as determined
in the experiment(s) of Example 1.
[0057] FIG. 9 illustrates that CRLF-2 specific protocells loaded
with the chemotherapeutic agent doxorubicin (DOX) induce apoptosis
of CRLF-2-positive cells (MHH CALL4) but not CRLF-2 negative cells
(MOLT4), as determined in the experiment(s) of Example 1.
[0058] FIGS. 10 and 11 depict data for selections against
BaF3/CRLF-2 (4.degree. C. as determined in the experiment(s) of
Example 1.
[0059] FIGS. 12 and 13 depict data for selections against
BaF3/CRLF-2 (37.degree. C.), as determined in the experiment(s) of
Example 1.
[0060] FIG. 14 depicts data for selections against BaF3/CRLF-2
(37.degree. C. with trypsin), as determined in the experiment(s) of
Example 1.
[0061] FIGS. 15(1)-15(8). Flow cytometry data of targeted and
non-targeted protocells to Baf3/CRLF2 and BaF3 parental cell lines,
as determined in the experiment(s) of Example 1. Particles were
labeled with Alexa-fluor-647 and incubated with various cell types
for an hour before the samples were washed and immediately measured
using a FACS Caliber Flow Cytometer. FIGS. 15(1) to 15(6) show
results for MTAAPVH-targeted protocells. FIGS. 15(7) and 15(8) show
GE-11 targeted protocells.
[0062] FIGS. 15(9-15(10). FIG. 15(9) shows the structure of a
plasmid that expresses the MS2 coat protein single-chain dimer with
a fusion of a CRLF2 targeting peptide (TDAHASV SEQ ID NO:7) at its
N-terminus. FIG. 15(10) shows the results of FACS analysis, which
reveals the ability of the targeted VLPs to specifically bind only
the cells producing CRLF2.
[0063] FIG. 16(a). CD19 IgG1 was partially reduced via reaction
with a 60-fold molar excess of TCEP for 20 minutes at room
temperature. Reduced antibody was then desalted and incubated with
protocells (DOPC with 30 wt % cholesterol and 10 wt %
maleimide-PEG-DMPE) overnight at 4 C. Protocells were washed
3.times. with PBS before being added to cells. Data determined in
the experiment of Example 2.
[0064] FIG. 16(b) shows that VLPs displaying anti-CD19 bind to
CD19-expressing NALM6 cells, but not CD19-negative Jurkat cells
(not shown). Data determined in the experiment of Example 2.
[0065] FIG. 17(a). Hierarchical Clustering Identifies 8 Cluster
Groups in High Risk ALL. Hierarchical clustering using 100 genes
(provided in Kang.sup.39) was used to identify clusters of patients
with shared patterns of gene expression. (Rows: Top 100 Probe Sets;
Columns: 207 ALL Patients). Shades of red depict expression levels
higher than the median while green indicates levels lower than the
median. The 8 cluster groups are outlined. Cases with an MLL
translocation are noted in yellow at the bottom of the figure while
cases with a t(1;19)(TCF3-PBX1) are noted in bright green. Cases
clustered in H2 that lacked a t(1;19)(TCF3-PBX1) are noted in dark
green. The red bars note patients who relapsed. Data determined in
the experiment of Example 3.
[0066] FIG. 17(b). Survival in Gene Expression Cluster Groups.
Relapse-free survival is shown for the patients in Cluster 8 (Panel
A), or those who express high levels of CRLF2 (Panel B) or C199
(Panel C). Red lines indicate the patients in the cluster or with
high gene expression while the black lines represent those either
in other cluster or with low levels of expression. Data determined
in the experiment of Example 3.
[0067] FIG. 18. Binding of M13 phage displaying a CRLF2-specific
peptide for BaF3-CRLF2 and BaF3 parental cells. Data determined in
the experiment of Example 4.
[0068] FIG. 19. CRLF2-targeted protocell binding/internalization by
CRLF2-positive cells (MUTZ5, MHHCALL4, BaF3/CRLF2) vs. controls
(BaF3 parental or NALM6 cells), as determined in the experiment(s)
of Example 5. A. CRLF2 targeting peptide density dependence of
dissociation constant K.sub.d. B. Confocal images of DOX
(fluorescent red) loaded protocells (silica; white) after
incubation with BaF3/CRLF2 or parental BaF3. C. Flow cytometric
binding of CRLF2-targeted protocells (loaded with DOX) after
binding and internalization in BaF3/CRLF2 and parental cells with
varying densities of octa-arginine (R8), which promotes
internalization.
[0069] FIG. 20 illustrates uptake of CRLF2-Targeted Protocells in
Established ALL Cell Lines (Mutz-5 and MHH CALL4) with High CRLF2
Cell Surface Expression vs. Controls (NALM-6). Left Panels: Cell
lines incubated with non-targeted protocells (top panels), then: 1)
CRLF2-targeted protocells after one hour at 4.degree. C., 2)
CRLF2-targeted protocells after one hour at 37.degree. C., and 3)
CRLF2-targeted protocells after 24 hours at 37.degree. C., imaged
using hyperspectral confocal fluorescence microscopy which detects
the encapsidated drug cargo (fluorescent doxorubicin (red), DOX in
each panel) as well as fluorescent silica cores (white). Right
Panels: Flow cytometric assays demonstrating intracellular uptake
of both DOX and the CRLF2-targeted protocells in 2-color flow
cytometric assays in CRLF2-expressing cell lines (Mutz-5, MHH
CALL4), but not control cells (NALM-6). Data determined in the
experiment(s) of Example 5.
[0070] FIG. 21 depicts fluorescent images of CRLF-2-expressing ALL
cells (MHH CALL4) and control cells (NALM6) that were continually
exposed to 75 nM of doxorubicin encapsidated within
CRLF-2-targeted, R8-modified protocells) for 48 hours at 37.degree.
C.), as determined in the experiment(s) of Example 5. MHH CALL4 and
not NALM6 cells demonstrate doxorubicin uptake and apoptosis
(annexin V) ALL-targeted protocell specificity and toxicity.
[0071] FIG. 22 illustrates live animal biophotonic imaging of
dye-loaded protocells (red, left panel), from 0 to 8 mg, 8 hours
after injection), as determined in the experiment(s) of Example 5.
These non-targeted protocells initially distributed widely
(protocells in the bladder are seen in the 2 mg mouse) and later
concentrated in the liver. Detection of dye-loaded protocells (red,
middle panel) in ALL-bearing mice (green, right panel). The CBG ALL
cells in the same mice are depicted in the middle and right
panels.
[0072] FIG. 23 illustrates the creation of CRLF2 (+) and (-) ALL
Models. A) Using lentiviral-mediated gene transfer, REH parental
cells were modified to express CBG and GFP plus/minus the CRLF2
gene), as determined in the experiment(s) of Example 5. Flow
cytometry demonstrates uniform expression of GFP and two separate
limiting dilution clones (3 and 4) over-expressing CRLF2. B)
Primary human ALL samples (see text) with or without CRLF2 &
JAK mutations. CRLF2 expression is detected by flow cytometry.
Below each set of histograms is in vivo imaging of the primary or
REH sample. Pseudocolor heat maps indicate presence of ALL on Day
20 (JH331, JL491, NL482b) or Day 3 (REH). No peripheral blasts are
detectable at these times.
[0073] FIG. 24 illustrates the non-specific toxicity of protocells
is a function of the charge of lipids employed in the SLB. (A) The
degree to which `empty` SP94-targeted protocells and liposomes, as
well as nanoporous silica cores induce oxidative stress and
subsequent cell death in Hep3B was determined using MitoSOX Red, a
mitochondrial superoxide indicator that fluoresces in the presence
of superoxide anions, and propidium iodide, respectively.
Positively- and negatively-charged polystyrene nanoparticles
(amine-PS and carboxyl-PS, respectively) were employed as positive
controls, while Hep3B exposed to 10 mM of the antioxidant,
N-acetylcysteine (NAC), was used as a negative control. All error
bars represent 95% confidence intervals (1.96.sigma.) for n=3. (B)
Confocal fluorescence microscopy of Hep3B cells exposed to DOPC or
DOTAP protocells for 24 hours at 37.degree. C. prior to being
stained with either MitoSOX Red or Alexa Fluor.RTM. 488-labeled
annexin V (green) and propidium iodide (red). Nuclei are stained
with DAPI. Scale bars=20 .mu.m. Data determined in the experiment
of Example 5.
[0074] FIG. 25 illustrates MS2 SP94 serum dilution versus OD-405,
as determined in the experiment(s) of Example 5.
[0075] FIG. 26 illustrates that combinations of peptides can be
used to direct targeting and internalization for non-internalized
receptors, as determined in the experiment(s) of Example 6.
[0076] FIGS. 27-29 depict the effect of 4 mg of fluorescently
labeled protocells in a murine luminescent leukemia model, as
determined in the experiment of Example 7.
[0077] FIG. 30 illustrates the MS2 VLP affinity selection process,
as described in the experiment of Example 8.
[0078] FIG. 31 depicts binding of M13 phage displaying a
CRLF2-specific peptide for BaF3-CRLF2 and BaF3 parental cells, as
determined in the experiment(s) of Example 9.
[0079] FIG. 32 depicts VLPs displaying anti-CD19
binding/CD19-expressing NALM6 cells, but not CD 19-negative Jurkat
cells (not shown), as determined in the experiment(s) of Example
9.
[0080] FIG. 33 illustrates protocell binding, internalization and
delivery, as determined in the experiment(s) of Example 9.
[0081] FIG. 34 illustrates that protocells modified with only six
SP94 peptides per particle exhibit a 10,000-fold greater affinity
for Hep3B than for normal hepatocytes, and other control cells, as
determined in the experiment(s) of Example 9.
[0082] FIG. 35 illustrates that CRLF2-targeted protocells were
demonstrated to possess a 1,000-fold higher affinity for engineered
BaF3-CRLF2 cells expressing high levels of CRLF2, as determined in
the experiment(s) of Example 9.
[0083] FIG. 36 illustrates uptake of CRLF2-targeted protocells in
established ALL cell lines (Mutz-5 and MHH CALL4) with high CRLF2
cell surface expression versus CRLF2-negative ALL cells (NALM-6),
as determined in the experiment(s) of Example 9.
[0084] FIG. 37 illustrates fluorescent images of CRLF2-expressing
ALL cells (MHH CALL4) and CRLF2-negative controls (NALM6) that were
continually exposed to 75 nM of doxorubicin encapsidated with
CRLF2-targeted, R8-modified protocells for 48 hours at 37.degree.
C.), as determined in the experiment(s) of Example 9.
[0085] FIG. 38 illustrates the impact of mTOR inhibition on four
high-risk ALL xenograft models representative of four different
CRLF2/JAK genotypes), as determined in the experiment(s) of Example
9.
[0086] FIG. 39 illustrates fluorescently tagged NALM6 cells that
were transduced with a retrovirus directing the expression of
ectopic human CRLF2 and stable clones with a 10-fold increase in
surface expression), as determined in the experiment(s) of Example
10.
DETAILED DESCRIPTION OF THE INVENTION
[0087] The following terms shall be used throughout the
specification to describe the present invention. Where a term is
not specifically defined herein, that term shall be understood to
be used in a manner consistent with its use by those of ordinary
skill in the art.
[0088] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention. In instances where a substituent is a
possibility in one or more Markush groups, it is understood that
only those substituents which form stable bonds are to be used.
[0089] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0090] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and" and "the" include plural
references unless the context clearly dictates otherwise.
[0091] Furthermore, the following terms shall have the definitions
set out below.
[0092] The term "patient" or "subject" is used throughout the
specification within context to describe an animal, generally a
mammal, especially including a domesticated animal and preferably a
human, to whom treatment, including prophylactic treatment
(prophylaxis), with the compounds or compositions according to the
present invention is provided. For treatment of those infections,
conditions or disease states which are specific for a specific
animal such as a human patient, the term patient refers to that
specific animal. In most instances, the patient or subject of the
present invention is a human patient of either or both genders.
[0093] The term "effective" is used herein, unless otherwise
indicated, to describe an amount of a compound or component which,
when used within the context of its use, produces or effects an
intended result, whether that result relates to the prophylaxis
and/or therapy of an infection and/or disease state or as otherwise
described herein. The term effective subsumes all other effective
amount or effective concentration terms (including the term
"therapeutically effective") which are otherwise described or used
in the present application.
[0094] The term "compound" is used herein to describe any specific
compound or bioactive agent disclosed herein, including any and all
stereoisomers (including diasteromers), individual optical isomers
(enantiomers) or racemic mixtures, pharmaceutically acceptable
salts and prodrug forms. The term compound herein refers to stable
compounds. Within its use in context, the term compound may refer
to a single compound or a mixture of compounds as otherwise
described herein.
[0095] The term "bioactive agent" refers to any biologically active
compound or drug which may be formulated for use in an embodiment
of the present invention. Exemplary bioactive agents include the
compounds according to the present invention which are used to
treat pediatric ALL or a disease state or condition which occurs
secondary to pediatric ALL and may include antiviral agents as well
as other compounds or agents which are otherwise described
herein.
[0096] The terms "treat", "treating", and "treatment", are used
synonymously to refer to any action providing a benefit to a
patient at risk for or afflicted with cancer, preferably pediatric
ALL, including improvement in the condition through lessening,
inhibition, suppression or elimination of at least one symptom,
delay in progression of pediatric ALL, prevention, delay in or
inhibition of the likelihood of the onset of pediatric ALL, etc. In
the case of viral infections associate with pediatric ALL, these
terms also apply to viral infections and preferably include, in
certain particularly favorable embodiments the eradication or
elimination (as provided by limits of diagnostics) of the virus
which is the causative agent of the infection.
[0097] Treatment, as used herein, encompasses both prophylactic and
therapeutic treatment of cancer, principally including pediatric
ALL, but also of other disease states associated with pediatric ALL
including viral infections. Compounds according to the present
invention can, for example, be administered prophylactically to a
mammal in advance of the occurrence of disease to reduce the
likelihood of that disease. Prophylactic administration is
effective to reduce or decrease the likelihood of the subsequent
occurrence of disease in the mammal, or decrease the severity of
disease (inhibition) that subsequently occurs, especially including
metastasis of cancer. Alternatively, compounds according to the
present invention can, for example, be administered therapeutically
to a mammal that is already afflicted by disease. In one embodiment
of therapeutic administration, administration of the present
compounds is effective to eliminate the disease and produce a
remission or substantially eliminate the likelihood of metastasis
of a cancer. Administration of the compounds according to the
present invention is effective to decrease the severity of the
disease or lengthen the lifespan of the mammal so afflicted, as in
the case of cancer, or inhibit or even eliminate the causative
agent of the disease, as in the case of viral co-infections.
[0098] The term "pharmaceutically acceptable" as used herein means
that the compound or composition is suitable for administration to
a subject, including a human patient, to achieve the treatments
described herein, without unduly deleterious side effects in light
of the severity of the disease and necessity of the treatment.
[0099] The term "inhibit" as used herein refers to the partial or
complete elimination of a potential effect, while inhibitors are
compounds/compositions that have the ability to inhibit.
[0100] The term "prevention" when used in context shall mean
"reducing the likelihood" or preventing a disease, condition or
disease state from occurring as a consequence of administration or
concurrent administration of one or more compounds or compositions
according to the present invention, alone or in combination with
another agent. It is noted that prophylaxis will rarely be 100%
effective; consequently the terms prevention and reducing the
likelihood are used to denote the fact that within a given
population of patients or subjects, administration with compounds
according to the present invention will reduce the likelihood or
inhibit a particular condition or disease state (in particular, the
worsening of a disease state such as the growth or metastasis of
cancer) or other accepted indicators of disease progression from
occurring.
[0101] The term "protocell" is used to describe a porous
nanoparticle which is made of a material comprising, e.g. silica,
polystyrene, alumina, titania, zirconia, or generally metal oxides,
organometallates, organosilicates or mixtures thereof.
[0102] In certain embodiments, the porous particle core may be
hydrophilic and can be further treated to provide a more
hydrophilic surface in order to influence pharmacological result in
a particular treatment modality. For example, mesoporous silica
particles according to the present invention can be further treated
with, for example, ammonium hydroxide or other bases and hydrogen
peroxide to provide significant hydrophilicity. The use of amine
containing silanes such as
3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEPTMS),
among others, may be used to produce negatively charged cores which
can markedly influence the cargo loading of the particles. Other
agents may be used to produce positively charged cores to influence
in the cargo in other instances, depending upon the physicochemical
characteristics of the cargo.
[0103] Nanoparticles according to the present invention comprise a
lipid bilayer which coats its surface to form a structure referred
to as a protocell. While numerous lipids and phospholipids may be
used to provide a lipid bilayer for use in the present invention,
in certain preferred embodiments, the lipid bilayer comprises a
phospholipid selected from the group consisting of phosphatidyl
choline, 1,2-Dioleoyl-3-Trimethylammonium-propane (DOTAP),
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)
or mixtures thereof. In addition to a phospholipid, including the
specific phospholipids as otherwise described herein, the lipid
bilayer may also comprise cholesterol (for structural integrity of
the lipid bilayer) as well as polyethylene glycol
lubricants/solvents (e.g. PEG 2000, etc.) to provide flexibility to
the lipid bilayer. In addition to fusing a single phospholipid
bilayer, multiple bilayers with opposite charges may be fused onto
the porous particles in order to further influence cargo loading,
sealing and release of the particle contents in a biological
system.
[0104] In certain embodiments, the lipid bilayer can be prepared,
for example, by extrusion of hydrated lipid films through a filter
of varying pore size (e.g., 50, 100, 200 nm) to provide filtered
lipid bilayer films, which can be fused with the porous particle
cores, for example, by pipette mixing or other standard method.
[0105] In various embodiments, the protocell (nanoparticle to which
a lipid bilayer covers or is otherwise fused to the particle) can
be loaded with and seal macromolecules (shRNAs, siRNAs, other micro
RNA and polypeptide toxins) as otherwise described herein, thus
creating a loaded protocell useful for cargo delivery across the
cell membrane
[0106] In preferred aspects of the present invention, the
protocells provide a targeted delivery through conjugation of
certain targeting peptides onto the protocell surface, preferably
by conjugation to the lipid bilayer surface. These peptides include
SP94 and H5WYG peptides which may be synthesized with C-terminal
cysteine residues and conjugated to one or more of the
phospholipids (especially, DOPE, which contains a
phosphoethanolamine group) which comprise the lipid bilayer or
conjugated to the phospholipids using one or more conjugating
agents.
[0107] The term "targeting peptide" is used to describe a preferred
targeting active species which is a peptide of a particular
sequence (preferably a 7mer as otherwise described herein, which
binds to a CRLF-2 receptor or other polypeptide in cancer cells and
allows the targeting of protocells according to the present
invention to particular cells which express a peptide (be it a
receptor or other functional polypeptide) to which the targeting
peptide binds. In the present invention, exemplary targeting
peptides include, for example, those which appear in FIGS. 3, and
10-14 hereof, and preferably include the following targeting
peptides: MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5), AAQTSTP
(SEQ ID NO:6), TDAHASV (SEQ ID NO:7), FSYLPSH (SEQ ID NO: 8),
YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ ID NO:10), AATLFPL (SEQ ID
NO:11), LTSRPTL (SEQ ID NO:12), ETKAWWL (SEQ ID NO:13), HWGMWSY
(SEQ ID NO:14), SQIFGNK (SEQ ID NO:15), SQAFVLV (SEQ ID NO:16),
WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID NO:18), TMCIYCT (SEQ ID
NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW (SEQ ID NO:21) and NILSLSM
(SEQ ID NO:22). Preferred CRLF-2 binding peptides include MTAAPVH
(SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID NO:6),
MHAPPFY (SEQ ID NO:10), ETKAWWL (SEQ ID NO:13), SQIFGNK (SEQ ID
NO:15), AATLFPL (SEQ ID NO:11), TDAHASV (SEQ ID NO:7) and FSYLPSH
(SEQ ID NO: 8). More preferably, the CRLF-2 binding peptide is
MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID
NO:6) or MHAPPFY (SEQ ID NO:10). Often, the CRLF-2 binding peptide
used in embodiments according to the present invention includes
MTAAPVH (SEQ ID NO: 4) and LTTPNWV (SEQ ID NO:5). Most often, the
CRLF-2 binding peptide is MTAAPVH (SEQ ID NO: 4).
[0108] Targeting peptides used herein are generally covalently
anchored/complexed to the phospholipic bilayer of VLPs as otherwise
described herein by conjugation through a crosslinking agent or by
complexing an appropriately modified peptide with an oligopeptide
such as hexameric histidine which can bind to copper and/or nickel
complexes of the phospholipid bilayer. Conjugation of peptides to
phospholipids represents a preferred approach for attaching
targeting peptides to protocells according to the present
invention. using crosslinking agents as otherwise described
herein.
[0109] Other targeting peptides are known in the art. Targeting
peptides may be complexed or preferably, covalently linked to the
lipid bilayer through use of a crosslinking agent as otherwise
described herein.
[0110] The term "crosslinking agent" is used to describe a
bifunctional compound of varying length containing two different
functional groups which may be used to covalently link various
components according to the present invention to each other.
Crosslinking agents according to the present invention may contain
two electrophilic groups (to react with nucleophilic groups on
peptides of oligonucleotides, one electrophilic group and one
nucleophilic group or two nucleophilic groups). The crosslinking
agents may vary in length depending upon the components to be
linked and the relative flexibility required. Crosslinking agents
are used to anchor targeting and/or fusogenic peptides to the
phospholipid bilayer, to link nuclear localization sequences to
histone proteins for packaging supercoiled plasmid DNA and in
certain instances, to crosslink lipids in the lipid bilayer of the
protocells. There are a large number of crosslinking agents which
may be used in the present invention, many commercially available
or available in the literature. Preferred crosslinking agents for
use in the present invention include, for example,
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),
N-[.beta.-Maleimidopropionic acid] hydrazide (BMPH),
NHS-(PEG).sub.n-maleimide,
succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester
(SM(PEG).sub.24), and succinimidyl
6-[3'-(2-pyridyldithio)-propionamido]hexanoate (LC-SPDP), among
others. Other crosslinking agents include, for example, AMAS, BMPS,
GMBS, sulfo-GMBS, MBS, sulfo-MBS, SMCC, sulfo-SMCC, EMCS,
sulfo-EMCS, SMPB, sulfo-SMPB, SMPH, LC-SMCC, Sulfo-KMUS,
SM(PEG)nNHS-PEG-Maleimide Crosslinkers, SPDP, LC-SPDP,
sulfo-LC-SPDP, SMPT, sulfo-LC-SMPT, SIA, SBAP, SIAB, or SIAB. These
crosslinkers are well known in the art. In some instances, it may
be advantageous to use crosslinkers which are cleavable via
reduction or at lower pH (selective for cancer cells). Using
cleavable crosslinkers helps to liberate cytotoxic agents in the
cytosol of target cells, e.g., cancer cells. Exemplary cleavable
crosslinking agents for use herein include SPDP, LC-SPDP,
sulfo-LC-SPDP, SMPT and sulf-LC-SMPT, among others.
[0111] The terms "fusogenic peptide" and "endosomolytic peptide"
are used synonymously to describe a peptide which is optionally and
preferred crosslinked onto the lipid bilayer surface of the
protocells or incorporated into the VLP according to the present
invention. Fusogenic peptides are incorporated onto protocells or
into VLPS in order to facilitate or assist escape from endosomal
bodies and to facilitate the introduction of protocells or VLPs
into targeted cells to effect an intended result (therapeutic
and/or diagnostic as otherwise described herein). Representative
and preferred fusogenic peptides for use in protocells according to
the present invention include H5WYG peptide, GLFHAIAHFIHGGWHGLIHGWY
(SEQ ID NO:24) modified for conjugation/crosslinking as
H.sub.2N-GLFHAIAHFIHGGWHGLIHGWYGGC-COOH (SEQ ID. NO: 3) or an 8 mer
polyarginine (H.sub.2N-RRRRRRRR-COOH, SEQ ID NO:23), modified for
conjugation/crosslinking as RRRRRRRRGGC, SEQ ID NO:2, among others
known in the art.
[0112] As used herein, unless otherwise specified, the term
"protocell" refers to a nanostructure having a porous particle and
a lipid bilayer surrounding the porous particle. The protocell can
mimic bioactive cells (or real cells) that have a supported lipid
bilayer membrane. For example, the porous particle can be made of a
material including polystyrene, silica, alumina, titania, zirconia,
etc. In embodiments, the porous particle 110 can have a
controllable average pore size ranging from about 2 nm to about 30
nm, and an average porosity ranging from about 10% to about 70%,
for example, ranging from about 25% to about 50%. The porous
particle can have an average particle size ranging from about 30 nm
to about 3000 nm.
[0113] The porous particle, such as porous silica particles, can be
surface charged. For example, the surface charge of the porous
silica particles can switch from negative to positive at neutral
pHs by using amine-modified silane precursors and controlling the
percentage of amine groups within the porous silica particles. For
example, the porous silica particles can have a composition of
about 5% to about 50% amine, such as about 10% to about 50% amine,
or about 5% to about 30% amine by weight; and the amine-modified
silane precursors can include, for example,
##STR00001##
[0114] The porous silica particles can be formed by, for example,
mixing water, HCl, ethanol, cetyltrimethylammonium bromide (CTAB),
and tetraethyl orthosilicate (TEOS), as disclosed in a related
International Patent Application No. PCT/US10/20096, entitled
"Porous Nanoparticle Supported Lipid Bilayer Nanostructures," which
is hereby incorporated by reference in its entirety.
[0115] Porous nanoparticulates used in protocells of the invention
include mesoporous silica nanoparticles and core-shell
nanoparticles.
[0116] The porous nanoparticulates can also be biodegradable
polymer nanoparticulates comprising one or more compositions
selected from the group consisting of aliphatic polyesters,
poly(lactic acid) (PLA), poly(glycolic acid) (PGA), co-polymers of
lactic acid and glycolic acid (PLGA), polycarprolactone (PCL),
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric
acid), poly(valeric acid), poly(lactide-co-caprolactone), alginate
and other polysaccharides, collagen, and chemical derivatives
thereof, albumin a hydrophilic protein, zein, a prolamine, a
hydrophobic protein, and copolymers and mixtures thereof.
[0117] A porous spherical silica nanoparticle is used for the
preferred protocells and is surrounded by a supported lipid or
polymer bilayer or multilayer. Various embodiments according to the
present invention provide nanostructures and methods for
constructing and using the nanostructures and providing protocells
according to the present invention. Many of the protocells in their
most elemental form are known in the art. Porous silica particles
of varying sizes ranging in size (diameter) from less than 5 nm to
200 nm or 500 nm or more are readily available in the art or can be
readily prepared using methods known in the art (see the examples
section) or alternatively, can be purchased from Melorium
Technologies, Rochester, N.Y. SkySpring Nanomaterials, Inc.,
Houston, Tex., USA or from Discovery Scientific, Inc., Vancouver,
British Columbia. Multimodal silica nanoparticles may be readily
prepared using the procedure of Carroll, et al., Langmuir, 25,
13540-13544 (2009). Protocells can be readily obtained using
methodologies known in the art. The examples section of the present
application provides certain methodology for obtaining protocells
which are useful in the present invention. Protocells according to
the present invention may be readily prepared, including protocells
comprising lipids which are fused to the surface of the silica
nanoparticle. See, for example, Liu, et al., Chem. Comm., 5100-5102
(2009), Liu, et al., J. Amer. Chem. Soc., 131, 1354-1355 (2009),
Liu, et al., J. Amer. Chem. Soc., 131, 7567-7569 (2009) Lu, et al.,
Nature, 398, 223-226 (1999), Preferred protocells for use in the
present invention are prepared according to the procedures which
are presented in Ashley, et al., Nature Materials, 2011, May;
10(5):389-97, Lu, et al., Nature, 398, 223-226 (1999), Caroll, et
al., Langmuir, 25, 13540-13544 (2009), and as otherwise presented
in the experimental section which follows.
[0118] The terms "nanoparticulate" and "porous nanoparticulate" are
used interchangeably herein and such particles may exist in a
crystalline phase, an amorphous phase, a semi-crystalline phase, a
semi amorphous phase, or a mixture thereof.
[0119] A nanoparticle may have a variety of shapes and
cross-sectional geometries that may depend, in part, upon the
process used to produce the particles. In one embodiment, a
nanoparticle may have a shape that is a sphere, a rod, a tube, a
flake, a fiber, a plate, a wire, a cube, or a whisker. A
nanoparticle may include particles having two or more of the
aforementioned shapes. In one embodiment, a cross-sectional
geometry of the particle may be one or more of circular,
ellipsoidal, triangular, rectangular, or polygonal. In one
embodiment, a nanoparticle may consist essentially of non-spherical
particles. For example, such particles may have the form of
ellipsoids, which may have all three principal axes of differing
lengths, or may be oblate or prelate ellipsoids of revolution.
Non-spherical nanoparticles alternatively may be laminar in form,
wherein laminar refers to particles in which the maximum dimension
along one axis is substantially less than the maximum dimension
along each of the other two axes. Non-spherical nanoparticles may
also have the shape of frusta of pyramids or cones, or of elongated
rods. In one embodiment, the nanoparticles may be irregular in
shape. In one embodiment, a plurality of nanoparticles may consist
essentially of spherical nanoparticles.
[0120] The phrase "effective average particle size" as used herein
to describe a multiparticulate (e.g., a porous nanoparticulate)
means that at least 50% of the particles therein are of a specified
size. Accordingly, "effective average particle size of less than
about 2,000 nm in diameter" means that at least 50% of the
particles therein are less than about 2000 nm in diameter. In
certain embodiments, nanoparticulates have an effective average
particle size of less than about 2,000 nm (i.e., 2 microns), less
than about 1,900 nm, less than about 1,800 nm, less than about
1,700 nm, less than about 1,600 nm, less than about 1,500 nm, less
than about 1,400 nm, less than about 1,300 nm, less than about
1,200 nm, less than about 1,100 nm, less than about 1,000 nm, less
than about 900 nm, less than about 800 nm, less than about 700 nm,
less than about 600 nm, less than about 500 nm, less than about 400
nm, less than about 300 nm, less than about 250 nm, less than about
200 nm, less than about 150 nm, less than about 100 nm, less than
about 75 nm, or less than about 50 nm, as measured by
light-scattering methods, microscopy, or other appropriate methods.
"D.sub.50" refers to the particle size below which 50% of the
particles in a multiparticulate fall. Similarly, "D.sub.90" is the
particle size below which 90% of the particles in a
multiparticulate fall.
[0121] In certain embodiments, the porous nanoparticulates are
comprised of one or more compositions selected from the group
consisting of silica, a biodegradable polymer, a solgel, a metal
and a metal oxide.
[0122] In an embodiment of the present invention, the
nanostructures include a core-shell structure which comprises a
porous particle core surrounded by a shell of lipid preferably a
bilayer, but possibly a monolayer or multilayer (see Liu, et al.,
JACS, 2009, Id). The porous particle core can include, for example,
a porous nanoparticle made of an inorganic and/or organic material
as set forth above surrounded by a lipid bilayer. In the present
invention, these lipid bilayer surrounded nanostructures are
referred to as "protocells" or "functional protocells," since they
have a supported lipid bilayer membrane structure. In embodiments
according to the present invention, the porous particle core of the
protocells can be loaded with various desired species ("cargo"),
including small molecules (e.g. anticancer agents as otherwise
described herein), large molecules (e.g. including macromolecules
such as RNA, including small interfering RNA or siRNA or small
hairpin RNA or shRNA, or other micro RNA or a polypeptide which may
include a polypeptide toxin such as a ricin toxin A-chain or other
toxic polypeptide such as diphtheria toxin A-chain DTx, cholera
toxin A-chain, among others) or a reporter polypeptide (e.g.
fluorescent green protein, among others) or semiconductor quantum
dots, or metallic nanoparticles, or metal oxide nanoparticles or
combinations thereof. In certain preferred aspects of the
invention, the protocells are loaded with super-coiled plasmid DNA,
which can be used to deliver a therapeutic and/or diagnostic
peptide(s) or a small hairpin RNA/shRNA, small interfering
RNA/siRNA or other micro RNA which can be used to inhibit
expression of proteins (such as, for example growth factor
receptors or other receptors which are responsible for or assist in
the growth of a cell especially a cancer cell, including epithelial
growth factor/EGFR, vascular endothelial growth factor
receptor/VEGFR-2 or platelet derived growth factor
receptor/PDGFR-.alpha., various cyclins as described hereinabove,
among numerous others, and induce growth arrest and apoptosis of
cancer cells).
[0123] In certain embodiments, the cargo components can include,
but are not limited to, chemical small molecules (especially
anticancer agents and antiviral agents, nucleic acids (DNA and RNA,
including siRNA, shRNA, other micro RNA and plasmids which, after
delivery to a cell, express one or more polypeptides or RNA
molecules), such as for a particular purpose, such as a therapeutic
application or a diagnostic application as otherwise disclosed
herein.
[0124] In embodiments, the lipid bilayer of the protocells can
provide biocompatibility and can be modified to possess targeting
species including, for example, targeting peptides including
antibodies, aptamers, and PEG (polyethylene glycol) to allow, for
example, further stability of the protocells and/or a targeted
delivery into a bioactive cell.
[0125] The protocells particle size distribution, according to the
present invention, depending on the application, may be
monodisperse or polydisperse. The silica cores can be rather
monodisperse (i.e., a uniform sized population varying no more than
about 5% in diameter e.g., .+-.10-nm for a 200 nm diameter
protocell especially if they are prepared using solution
techniques) or rather polydisperse (i.e., a polydisperse population
can vary widely from a mean or medium diameter, e.g., up to
.+-.200-nm or more if prepared by aerosol. See FIG. 1, attached.
Polydisperse populations can be sized into monodisperse
populations. All of these are suitable for protocell formation. In
the present invention, preferred protocells are preferably no more
than about 500 nm in diameter, preferably no more than about 200 nm
in diameter in order to afford delivery to a patient or subject and
produce an intended therapeutic effect.
[0126] In one embodiment, the present invention is directed to high
surface area (i.e., greater than about 600 m.sup.2/g, preferably
about 600 to about 1,000-1,250 mg.sup.2/g), preferably monodisperse
spherical silica or other biocompatible material nanoparticles
having diameters falling within the range of about 0.05 to 50
.mu.m, preferably about 1,000 nm or less, more preferably about 100
nm or less, 10-20 nm in diameter, a multimodal pore morphology
comprising large (about 1-100 nm, preferably about 2-50 nm, more
preferably about 10-35 nm, about 20-30 nm) surface-accessible pores
interconnected by smaller internal pores (about 2-20 nm, preferably
about 5-15 nm, more preferably about 6-12 nm) volume, each
nanoparticle comprising a lipid bilayer (preferably a phospholipid
bilayer) supported by said nanoparticles (the phospholipic bilayer
and silica nanoparticles together are labeled "protocells"), to
which is bound at least one antigen which binds to a targeting
polypeptide or protein on a cell to which the protocells are to be
targeted, wherein the protocells further comprise (are loaded) with
a small molecule anticancer agent and/or a macromolecule selected
from the group consisting of a short hairpin RNA (shRNA), a small
interfering RNA (siRNA) or a polypeptide toxin (e.g. ricin toxin
A-chain or other toxic polypeptide).
[0127] The term "monodisperse" is used as a standard definition
established by the National Institute of Standards and Technology
(NIST) (Particle Size Characterization, Special Publication 960-1,
January 2001) to describe a distribution of particle size within a
population of particles, in this case nanoparticles, which particle
distribution may be considered monodisperse if at least 90% of the
distribution lies within 5% of the median size. See Takeuchi, et
al., Advanced Materials, 2005, 17, No. 8, 1067-1072. In certain
embodiments, protocells according to the present invention utilize
nanoparticles to form protocells which are monodisperse.
[0128] In certain embodiments, protocells according to the present
invention generally range in size from greater than about 8-10 nm
to about 5 .mu.m in diameter, preferably about 20-nm-3 .mu.m in
diameter, about 10 nm to about 500 nm, more preferably about
20-200-nm (including about 150 nm, which may be a mean or median
diameter). As discussed above, the protocell population may be
considered monodisperse or polydisperse based upon the mean or
median diameter of the population of protocells. Size is very
important to therapeutic and diagnostic aspects of the present
invention as particles smaller than about 8-nm diameter are
excreted through kidneys, and those particles larger than about 200
nm are trapped by the liver and spleen. Thus, an embodiment of the
present invention focuses in smaller sized protocells for drug
delivery and diagnostics in the patient or subject.
[0129] In certain embodiments, protocells according the present
invention are characterized by containing mesopores, preferably
pores which are found in the nanostructure material. These pores
(at least one, but often a large plurality) may be found
intersecting the surface of the nanoparticle (by having one or both
ends of the pore appearing on the surface of the nanoparticle) or
internal to the nanostructure with at least one or more mesopore
interconnecting with the surface mesopores of the nanoparticle.
Interconnecting pores of smaller size are often found internal to
the surface mesopores. The overall range of pore size of the
mesopores can be 0.03-50-nm in diameter. Preferred pore sizes of
mesopores range from about 2-30 nm; they can be monosized or
bimodal or graded--they can be ordered or disordered (essentially
randomly disposed or worm-like). See FIG. 2, attached.
[0130] Mesopores (IUPAC definition 2-50-nm in diameter) are
`molded` by templating agents including surfactants, block
copolymers, molecules, macromolecules, emulsions, latex beads, or
nanoparticles. In addition, processes could also lead to micropores
(IUPAC definition less than 2-nm in diameter) all the way down to
about 0.03-nm e.g. if a templating moiety in the aerosol process is
not used. They could also be enlarged to macropores, i.e., 50-nm in
diameter.
[0131] Pore surface chemistry of the nanoparticle material can be
very diverse--all organosilanes yielding cationic, anionic,
hydrophilic, hydrophobic, reactive groups--pore surface chemistry,
especially charge and hydrophobicity, affect loading capacity. See
FIG. 3, attached. Attractive electrostatic interactions or
hydrophobic interactions control/enhance loading capacity and
control release rates. Higher surface areas can lead to higher
loadings of drugs/cargos through these attractive interactions. See
below.
[0132] In certain embodiments, the surface area of nanoparticles,
as measured by the N2 BET method, ranges from about 100 m2/g to
>about 1200 m2/g. In general, the larger the pore size, the
smaller the surface area. See table FIG. 2A. The surface area
theoretically could be reduced to essentially zero, if one does not
remove the templating agent or if the pores are sub-0.5-nm and
therefore not measurable by N2 sorption at 77K due to kinetic
effects. However, in this case, they could be measured by CO2 or
water sorption, but would probably be considered non-porous. This
would apply if biomolecules are encapsulated directly in the silica
cores prepared without templates, in which case particles (internal
cargo) would be released by dissolution of the silica matrix after
delivery to the cell.
[0133] Typically the protocells according to the present invention
are loaded with cargo to a capacity up to about 50 weight %:
defined as (cargo weight/weight of loaded protocell).times.100. The
optimal loading of cargo is often about 0.01 to 10% but this
depends on the drug or drug combination which is incorporated as
cargo into the protocell. This is generally expressed in .mu.M per
10.sup.10 particles where we have values ranging from 2000-100
.mu.M per 10.sup.10 particles. Preferred protocells according to
the present invention exhibit release of cargo at pH about 5.5,
which is that of the endosome, but are stable at physiological pH
of 7 or higher (7.4).
[0134] The surface area of the internal space for loading is the
pore volume whose optimal value ranges from about 1.1 to 0.5 cubic
centimeters per gram (cc/g). Note that in the protocells according
to one embodiment of the present invention, the surface area is
mainly internal as opposed to the external geometric surface area
of the nanoparticle.
[0135] The lipid bilayer supported on the porous particle according
to one embodiment of the present invention has a lower melting
transition temperature, i.e. is more fluid than a lipid bilayer
supported on a non-porous support or the lipid bilayer in a
liposome. This is sometimes important in achieving high affinity
binding of targeting ligands at low peptide densities, as it is the
bilayer fluidity that allows lateral diffusion and recruitment of
peptides by target cell surface receptors. One embodiment provides
for peptides to cluster, which facilitates binding to a
complementary target.
[0136] In the present invention, the lipid bilayer may vary
significantly in composition. Ordinarily, any lipid or polymer
which is may be used in liposomes may also be used in protocells.
Preferred lipids are as otherwise described herein. Particularly
preferred lipid bilayers for use in protocells according to the
present invention comprise a mixtures of lipids (as otherwise
described herein) at a weight ratio of 5% DOPE, 5% PEG, 30%
cholesterol, 60% DOPC or DPPC (by weight).
[0137] The charge of the mesoporous silica NP core as measured by
the Zeta potential may be varied monotonically from -50 to +50 mV
by modification with the amine silane, 2-(aminoethyl)
propyltrimethoxy-silane (AEPTMS) or other organosilanes. This
charge modification, in turn, varies the loading of the drug within
the cargo of the protocell. Generally, after fusion of the
supported lipid bilayer, the zeta-potential is reduced to between
about -10 mV and +5 mV, which is important for maximizing
circulation time in the blood and avoiding non-specific
interactions.
[0138] Depending on how the surfactant template is removed, e.g.
calcination at high temperature (500.degree. C.) versus extraction
in acidic ethanol, and on the amount of AEPTMS incorporated in the
silica framework, the silica dissolution rates can be varied
widely. This in turn controls the release rate of the internal
cargo. This occurs because molecules that are strongly attracted to
the internal surface area of the pores diffuse slowly out of the
particle cores, so dissolution of the particle cores controls in
part the release rate.
[0139] Further characteristics of protocells according to an
embodiment of the present invention are that they are stable at pH
7, i.e. they don't leak their cargo, but at pH 5.5, which is that
of the endosome lipid or polymer coating becomes destabilized
initiating cargo release. This pH-triggered release is important
for maintaining stability of the protocell up until the point that
it is internalized in the cell by endocytosis, whereupon several pH
triggered events cause release into the endosome and consequently,
the cytosol of the cell. Quantitative experimental evidence has
shown that targeted protocells illicit only a weak immune response,
because they do not support T-Cell help required for higher
affinity IgG, a favorable result.
[0140] Protocells according to the present invention exhibit at
least one or more a number of characteristics (depending upon the
embodiment) which distinguish them from prior art protocells:
[0141] 1) The protocells target CRLF-2 and/or CD 19 and in contrast
to the prior art, an embodiment of the present invention specifies
nanoparticles whose average size (diameter) is less than about
200-nm--this size is engineered to enable efficient cellular uptake
by receptor mediated endocytosis; [0142] 2) An embodiment of the
present invention targets CRLF-2 and/or CD 19 and can specify both
monodisperse and/or polydisperse sizes to enable control of
biodistribution. [0143] 3) An embodiment of the present invention
is directed to nanoparticles that target CRLF-2 and/or CD19 and
that induce receptor mediated endocytosis. [0144] 4) An embodiment
of the present invention targets CRLF-2 and/or CD 19 and induces
dispersion of cargo into cytoplasm through the inclusion of
fusogenic or endosomolytic peptides. [0145] 5) An embodiment of the
present invention targets CRLF-2 and/or CD19 and provides particles
with pH triggered release of cargo. [0146] 6) An embodiment of the
present invention targets CRLF-2 and/or CD19 and exhibits
controlled time dependent release of cargo (via extent of thermally
induced crosslinking of silica nanoparticle matrix). [0147] 7) An
embodiment of the present invention targets CRLF-2 and/or CD19 and
can exhibit time dependent pH triggered release. [0148] 8) An
embodiment of the present invention targets CRLF-2 and/or CD19 and
can contain and provide cellular delivery of complex multiple
cargoes. [0149] 9) An embodiment of the present invention shows the
killing of CRLF-2 and/or CD19-expressing cancer cells. [0150] 10)
An embodiment of the present invention shows diagnosis of CRLF-2
and/or CD19-expressing cancer cells. [0151] 11) An embodiment of
the present invention shows selective entry of target cells. [0152]
12) An embodiment of the present invention shows selective
exclusion from off-target cells (selectivity). [0153] 13) An
embodiment of the present invention targets CRLF-2 and/or CD
19-expressing cancer cells and shows enhanced fluidity of the
supported lipid bilayer. [0154] 14) An embodiment of the present
invention targets CRLF-2 and/or CD19-expressing cancer cells and
exhibits sub-nanomolar and controlled binding affinity to target
cells. [0155] 15) An embodiment of the present invention exhibits
sub-nanomolar binding affinity to CRLF-2 and/or CD19-expressing
cancer cells and also exhibits targeting ligand densities below
concentrations found in the prior art. [0156] 16) An embodiment of
the present invention can further distinguish the prior art with
with finer levels of detail unavailable in the prior art.
[0157] The term "lipid" is used to describe the components which
are used to form lipid bilayers on the surface of the nanoparticles
which are used in the present invention. Various embodiments
provide nanostructures which are constructed from nanoparticles
which support a lipid bilayer(s). In embodiments according to the
present invention, the nanostructures preferably include, for
example, a core-shell structure including a porous particle core
surrounded by a shell of lipid bilayer(s). The nanostructure,
preferably a porous silica nanostructure as described above,
supports the lipid bilayer membrane structure. In embodiments
according to the invention, the lipid bilayer of the protocells can
provide biocompatibility and can be modified to possess targeting
species including, for example, targeting peptides, fusogenic
peptides, antibodies, aptamers, and PEG (polyethylene glycol) to
allow, for example, further stability of the protocells and/or a
targeted delivery into a bioactive cell, in particular a cancer
cell. PEG, when included in lipid bilayers, can vary widely in
molecular weight (although PEG ranging from about 10 to about 100
units of ethylene glycol, about 15 to about 50 units, about 15 to
about 20 units, about 15 to about 25 units, about 16 to about 18
units, etc, may be used and the PEG component which is generally
conjugated to phospholipid through an amine group comprises about
1% to about 20%, preferably about 5% to about 15%, about 10% by
weight of the lipids which are included in the lipid bilayer.
[0158] Numerous lipids which are used in liposome delivery systems
may be used to form the lipid bilayer on nanoparticles to provide
protocells according to the present invention. Virtually any lipid
which is used to form a liposome may be used in the lipid bilayer
which surrounds the nanoparticles to form protocells according to
an embodiment of the present invention. Preferred lipids for use in
the present invention include, for example,
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof. Cholesterol, not technically a
lipid, but presented as a lipid for purposes of an embodiment of
the present invention given the fact that cholesterol may be an
important component of the lipid bilayer of protocells according to
an embodiment of the invention. Often cholesterol is incorporated
into lipid bilayers of protocells in order to enhance structural
integrity of the bilayer. These lipids are all readily available
commercially from Avanti Polar Lipids, Inc. (Alabaster, Ala., USA).
DOPE and DPPE are particularly useful for conjugating (through an
appropriate crosslinker) peptides, polypeptides, including
antibodies, RNA and DNA through the amine group on the lipid.
[0159] In certain embodiments, the porous nanoparticulates can also
be biodegradable polymer nanoparticulates comprising one or more
compositions selected from the group consisting of aliphatic
polyesters, poly(lactic acid) (PLA), poly(glycolic acid) (PGA),
co-polymers of lactic acid and glycolic acid (PLGA),
polycarprolactone (PCL), polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butyric acid), poly(valeric acid),
poly(lactide-co-caprolactone), alginate and other polysaccharides,
collagen, and chemical derivatives thereof, albumin a hydrophilic
protein, zein, a prolamine, a hydrophobic protein, and copolymers
and mixtures thereof.
[0160] In still other embodiments, the porous nanoparticles each
comprise a core having a core surface that is essentially free of
silica, and a shell attached to the core surface, wherein the core
comprises a transition metal compound selected from the group
consisting of oxides, carbides, sulfides, nitrides, phosphides,
borides, halides, selenides, tellurides, tantalum oxide, iron oxide
or combinations thereof.
[0161] The silica nanoparticles used in the present invention can
be, for example, mesoporous silica nanoparticles and core-shell
nanoparticles. The nanoparticles may incorporate an absorbing
molecule, e.g. an absorbing dye. Under appropriate conditions, the
nanoparticles emit electromagnetic radiation resulting from
chemiluminescence.
[0162] Mesoporous silica nanoparticles can be e.g. from around 5 nm
to around 500 nm in size, including all integers and ranges there
between. The size is measured as the longest axis of the particle.
In various embodiments, the particles are from around 10 nm to
around 500 nm and from around 10 nm to around 100 nm in size. The
mesoporous silica nanoparticles have a porous structure. The pores
can be from around 1 to around 20 nm in diameter, including all
integers and ranges there between. In one embodiment, the pores are
from around 1 to around 10 nm in diameter. In one embodiment,
around 90% of the pores are from around 1 to around 20 nm in
diameter. In another embodiment, around 95% of the pores are around
1 to around 20 nm in diameter.
[0163] The mesoporous nanoparticles can be synthesized according to
methods known in the art. In one embodiment, the nanoparticles are
synthesized using sol-gel methodology where a silica precursor or
silica precursors and a silica precursor or silica precursors
conjugated (i.e., covalently bound) to absorber molecules are
hydrolyzed in the presence of templates in the form of micelles.
The templates are formed using a surfactant such as, for example,
hexadecyltrimethylammonium bromide (CTAB). It is expected that any
surfactant which can form micelles can be used.
[0164] The core-shell nanoparticles comprise a core and shell. The
core comprises silica and an absorber molecule. The absorber
molecule is incorporated in to the silica network via a covalent
bond or bonds between the molecule and silica network. The shell
comprises silica.
[0165] In one embodiment, the core is independently synthesized
using known sol-gel chemistry, e.g., by hydrolysis of a silica
precursor or precursors. The silica precursors are present as a
mixture of a silica precursor and a silica precursor conjugated,
e.g., linked by a covalent bond, to an absorber molecule (referred
to herein as a "conjugated silica precursor"). Hydrolysis can be
carried out under alkaline (basic) conditions to form a silica core
and/or silica shell. For example, the hydrolysis can be carried out
by addition of ammonium hydroxide to the mixture comprising silica
precursor(s) and conjugated silica precursor(s).
[0166] Silica precursors are compounds which under hydrolysis
conditions can form silica. Examples of silica precursors include,
but are not limited to, organosilanes such as, for example,
tetraethoxysilane (TEOS), tetramethoxysilane (TMOS) and the
like.
[0167] The silica precursor used to form the conjugated silica
precursor has a functional group or groups which can react with the
absorbing molecule or molecules to form a covalent bond or bonds.
Examples of such silica precursors include, but is not limited to,
isocyanatopropyltriethoxysilane (ICPTS),
aminopropyltrimethoxysilane (APTS), mercaptopropyltrimethoxysilane
(MPTS), and the like.
[0168] In one embodiment, an organosilane (conjugatable silica
precursor) used for forming the core has the general formula
R.sub.4nSiX.sub.n, where X is a hydrolyzable group such as ethoxy,
methoxy, or 2-methoxy-ethoxy; R can be a monovalent organic group
of from 1 to 12 carbon atoms which can optionally contain, but is
not limited to, a functional organic group such as mercapto, epoxy,
acrylyl, methacrylyl, or amino; and n is an integer of from 0 to 4.
The conjugatable silica precursor is conjugated to an absorber
molecule and subsequently co-condensed for forming the core with
silica precursors such as, for example, TEOS and TMOS. A silane
used for forming the silica shell has n equal to 4. The use of
functional mono-, bis- and tris-alkoxysilanes for coupling and
modification of co-reactive functional groups or hydroxy-functional
surfaces, including glass surfaces, is also known, see Kirk-Othmer,
Encyclopedia of Chemical Technology, Vol. 20, 3rd Ed., J. Wiley,
N.Y.; see also E. Pluedemann, Silane Coupling Agents, Plenum Press,
N.Y. 1982. The organo-silane can cause gels, so it may be desirable
to employ an alcohol or other known stabilizers. Processes to
synthesize core-shell nanoparticles using modified Stoeber
processes can be found in U.S. patent application Ser. Nos.
10/306,614 and 10/536, 569, the disclosure of such processes
therein are incorporated herein by reference.
[0169] "Amine-containing silanes" include, but are not limited to,
a primary amine, a secondary amine or a tertiary amine
functionalized with a silicon atom, and may be a monoamine or a
polyamine such as diamine. Preferably, the amine-containing silane
is N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEPTMS).
Non-limiting examples of amine-containing silanes also include
3-aminopropyltrimethoxysilane (APTMS) and
3-aminopropyltriethoxysilane (APTS), as well as an amino-functional
trialkoxysilane. Protonated secondary amines, protonated tertiary
alkyl amines, protonated amidines, protonated guanidines,
protonated pyridines, protonated pyrimidines, protonated pyrazines,
protonated purines, protonated imidazoles, protonated pyrroles,
quaternary alkyl amines, or combinations thereof, can also be
used.
[0170] In certain embodiments of a protocell of the invention, the
lipid bilayer is comprised of one or more lipids selected from the
group consisting of phosphatidyl-cholines (PCs) and
cholesterol.
[0171] In certain embodiments, the lipid bilayer is comprised of
one or more phosphatidyl-cholines (PCs) selected from the group
consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), egg PC,
and a lipid mixture comprising between about 50% to about 70%, or
about 51% to about 69%, or about 52% to about 68%, or about 53% to
about 67%, or about 54% to about 66%, or about 55% to about 65%, or
about 56% to about 64%, or about 57% to about 63%, or about 58% to
about 62%, or about 59% to about 61%, or about 60%, of one or more
unsaturated phosphatidyl-cholines, DMPC [14:0] having a carbon
length of 14 and no unsaturated bonds,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) [16:0],
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) [18:0],
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) [18:1
(.DELTA.9-Cis)], POPC [16:0-18:1], and DOTAP [18:1].
[0172] In other embodiments:
(a) the lipid bilayer is comprised of a mixture of (1) egg PC, and
(2) one or more phosphatidyl-cholines (PCs) selected from the group
consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a lipid
mixture comprising between about 50% to about 70% or about 51% to
about 69%, or about 52% to about 68%, or about 53% to about 67%, or
about 54% to about 66%, or about 55% to about 65%, or about 56% to
about 64%, or about 57% to about 63%, or about 58% to about 62%, or
about 59% to about 61%, or about 60%, of one or more unsaturated
phosphatidyl-choline, DMPC [14:0] having a carbon length of 14 and
no unsaturated bonds, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) [16:0], 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)
[18:0], 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) [18:1
(A9-Cis)], POPC [16:0-18:1] and DOTAP [18:1]; and wherein (b) the
molar concentration of egg PC in the mixture is between about 10%
to about 50% or about 11% to about 49%, or about 12% to about 48%,
or about 13% to about 47%, or about 14% to about 46%, or about 15%
to about 45%, or about 16% to about 44%, or about 17% to about 43%,
or about 18% to about 42%, or about 19% to about 41%, or about 20%
to about 40%, or about 21% to about 39%, or about 22% to about 38%,
or about 23% to about 37%, or about 24% to about 36%, or about 25%
to about 35%, or about 26% to about 34%, or about 27% to about 33%,
or about 28% to about 32%, or about 29% to about 31%, or about
30%.
[0173] In certain embodiments, the lipid bilayer is comprised of
one or more compositions selected from the group consisting of a
phospholipid, a phosphatidyl-choline, a phosphatidyl-serine, a
phosphatidyl-diethanolamine, a phosphatidylinosite, a sphingolipid,
and an ethoxylated sterol, or mixtures thereof. In illustrative
examples of such embodiments, the phospholipid can be a lecithin;
the phosphatidylinosite can be derived from soy, rape, cotton seed,
egg and mixtures thereof; the sphingolipid can be ceramide, a
cerebroside, a sphingosine, and a sphingomyelin, and a mixture
thereof; the ethoxylated sterol can be phytosterol,
PEG-(polyethyleneglykol)-5-soy bean sterol, and
PEG-(polyethyleneglykol)-5 rapeseed sterol. In certain embodiments,
the phytosterol comprises a mixture of at least two of the
following compositions: sistosterol, camposterol and
stigmasterol.
[0174] In still other illustrative embodiments, the lipid bilayer
is comprised of one or more phosphatidyl groups selected from the
group consisting of phosphatidyl choline,
phosphatidyl-ethanolamine, phosphatidyl-serine,
phosphatidyl-inositol, lyso-phosphatidyl-choline,
lyso-phosphatidyl-ethanolamnine, lyso-phosphatidyl-inositol and
lyso-phosphatidyl-inositol.
[0175] In still other illustrative embodiments, the lipid bilayer
is comprised of phospholipid selected from a monoacyl or
diacylphosphoglyceride.
[0176] In still other illustrative embodiments, the lipid bilayer
is comprised of one or more phosphoinositides selected from the
group consisting of phosphatidyl-inositol-3-phosphate (PI-3-P),
phosphatidyl-inositol-4-phosphate (PI-4-P),
phosphatidyl-inositol-5-phosphate (PT-5-P),
phosphatidyl-inositol-3,4-diphosphate (PI-3,4-P2),
phosphatidyl-inositol-3,5-diphosphate (PI-3,5-P2),
phosphatidyl-inositol-4,5-diphosphate (PI-4,5-P2),
phosphatidyl-inositol-3,4,5-triphosphate (PI-3,4,5-P3),
lysophosphatidyl-inositol-3-phosphate (LPI-3-P),
lysophosphatidyl-inositol-4-phosphate (LPI-4-P),
lysophosphatidyl-inositol-5-phosphate (LPI-5-P),
lysophosphatidyl-inositol-3,4-diphosphate (LPI-3,4-P2),
lysophosphatidyl-inositol-3,5-diphosphate (LPI-3,5-P2),
lysophosphatidyl-inositol-4,5-diphosphate (LPI-4,5-P2), and
lysophosphatidyl-inositol-3,4,5-triphosphate (LPI-3,4,5-P3), and
phosphatidyl-inositol (PI), and lysophosphatidyl-inositol
(LPI).
[0177] In still other illustrative embodiments, the lipid bilayer
is comprised of one or more phospholipids selected from the group
consisting of PEG-poly(ethylene glycol)-derivatized
distearoylphosphatidylethanolamine (PEG-DSPE), poly(ethylene
glycol)-derivatized ceramides (PEG-CER), hydrogenated soy
phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC),
phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG),
phosphatidyl insitol (PI), monosialogangolioside, spingomyelin
(SPM), distearoylphosphatidylcholine (DSPC),
dimyristoylphosphatidylcholine (DMPC), and
dimyristoylphosphatidylglycerol (DMPG).
[0178] In one illustrative embodiment of a protocell of the
invention:
(a) include at least one anticancer agent that targets CRLF-2
and/or CD 19-expressing cancer cells and that is effective in the
treatment of pediatric ALL, including B-Cell ALL; (b) less than
around 10% to around 20% of the anticancer agent is released from
the porous nanoparticulates in the absence of a reactive oxygen
species; and (c) upon disruption of the lipid bilayer as a result
of contact with a reactive oxygen species, the porous
nanoparticulates release an amount of anticancer agent that is
approximately equal to around 60% to around 80%, or around 61% to
around 79%, or around 62% to around 78%, or around 63% to around
77%, or around 64% to around 77%, or around 65% to around 76%, or
around 66% to around 75%, or around 67% to around 74%, or around
68% to around 73%, or around 69% to around 72%, or around 70% to
around 71%, or around 70% of the amount of anticancer agent that
would have been released had the lipid bilayer been lysed with 5%
(w/v) Triton X-100.
[0179] One illustrative embodiment of a protocell of the invention
includes at least one anticancer agent that targets CRLF-2 and/or
CD 19-expressing cancer cells, is effective in the treatment of ALL
and that comprises a plurality of negatively-charged, nanoporous,
nanoparticulate silica cores that:
(a) are modified with an amine-containing silane selected from the
group consisting of (1) a primary amine, a secondary amine a
tertiary amine, each of which is functionalized with a silicon atom
(2) a monoamine or a polyamine (3)
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEPTMS) (4)
3-aminopropyltrimethoxysilane (APTMS) (5)
3-aminopropyltriethoxysilane (APTS) (6) an amino-functional
trialkoxysilane, and (7) protonated secondary amines, protonated
tertiary alkyl amines, protonated amidines, protonated guanidines,
protonated pyridines, protonated pyrimidines, protonated pyrazines,
protonated purines, protonated imidazoles, protonated pyrroles, and
quaternary alkyl amines, or combinations thereof; (b) are loaded
with a shRNA, siRNA or ricin toxin A-chain or mixtures thereof; and
(c) that are encapsulated by and that support a lipid bilayer
comprising one of more lipids selected from the group consisting of
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine] (DOPS),
1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (18:1 PEG-2000 PE),
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (16:0 PEG-2000 PE),
1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-Glyce-
ro-3-Phosphocholine (18:1-12:0 NBD PC),
1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-gl-
ycero-3-phosphocholine (16:0-12:0 NBD PC), cholesterol and
mixtures/combinations thereof; and wherein the lipid bilayer
comprises a cationic lipid and one or more zwitterionic
phospholipids.
[0180] Protocells of the invention can comprise a wide variety of
pharmaceutically-active ingredients in addition to anticancer
agents that target CRLF-2 and/or CD19-expressing cancer cells and
that are effective in the treatment of cancer, including pediatric
ALL.
[0181] As used herein, the term "polynucleotide" refers to a
polymeric form of nucleotides of any length, either ribonucleotides
or deoxynucleotides, and includes both double- and single-stranded
DNA and RNA. A polynucleotide may include nucleotide sequences
having different functions, such as coding regions, and non-coding
regions such as regulatory sequences (e.g., promoters or
transcriptional terminators). A polynucleotide can be obtained
directly from a natural source, or can be prepared with the aid of
recombinant, enzymatic, or chemical techniques. A polynucleotide
can be linear or circular in topology. A polynucleotide can be, for
example, a portion of a vector, such as an expression or cloning
vector, or a fragment.
[0182] As used herein, the term "polypeptide" refers broadly to a
polymer of two or more amino acids joined together by peptide
bonds. The term "polypeptide" also includes molecules which contain
more than one polypeptide joined by a disulfide bond, or complexes
of polypeptides that are joined together, covalently or
noncovalently, as multimers (e g., dimers, tetramers). Thus, the
terms peptide, oligopeptide, and protein are all included within
the definition of polypeptide and these terms are used
interchangeably. It should be understood that these terms do not
connote a specific length of a polymer of amino acids, nor are they
intended to imply or distinguish whether the polypeptide is
produced using recombinant techniques, chemical or enzymatic
synthesis, or is naturally occurring.
[0183] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the desired functional is retained by the polypeptide. NH.sub.2
refers to the free amino group present at the amino terminus of a
polypeptide. COOH refers to the free carboxy group present at the
carboxy terminus of a polypeptide.
[0184] The term "coding sequence" is defined herein as a portion of
a nucleic acid sequence which directly specifies the amino acid
sequence of its protein product. The boundaries of the coding
sequence are generally determined by a ribosome binding site
(prokaryotes) or by the ATG start codon (eukaryotes) located just
upstream of the open reading frame at the 5'-end of the mRNA and a
transcription terminator sequence located just downstream of the
open reading frame at the 3'-end of the mRNA. A coding sequence can
include, but is not limited to, DNA, cDNA, and recombinant nucleic
acid sequences.
[0185] A "heterologous" region of a recombinant cell is an
identifiable segment of nucleic acid within a larger nucleic acid
molecule that is not found in association with the larger molecule
in nature.
[0186] An "origin of replication" refers to those DNA sequences
that participate in DNA synthesis.
[0187] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation, as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase. Eukaryotic promoters will often, but not always,
contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain
Shine-Dalgarno sequences in addition to the -10 and -35 consensus
sequences.
[0188] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "under the control" of
transcriptional and translational control sequences in a cell when
RNA polymerase transcribes the coding sequence into mRNA, which is
then translated into the protein encoded by the coding sequence.
Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0189] A "signal sequence" can be included before the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates to the host cell to direct the
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell
before the protein leaves the cell.
Signal sequences can be found associated with a variety of proteins
native to prokaryotes and eukaryotes.
[0190] A cell has been "transformed" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. The
transforming DNA may or may not be integrated (covalently linked)
into chromosomal DNA making up the genome of the cell. In
prokaryotes, yeast, and mammalian cells for example, the
transforming DNA may be maintained on an episomal element such as a
plasmid. With respect to eukaryotic cells, a stably transformed
cell is one in which the transforming DNA has become integrated
into a chromosome so that it is inherited by daughter cells through
chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones
comprised of a population of daughter cells containing the
transforming DNA.
[0191] It should be appreciated that also within the scope of the
present invention are nucleic acid sequences encoding the
polypeptide(s) of the present invention, which code for a
polypeptide having the same amino acid sequence as the sequences
disclosed herein, but which are degenerate to the nucleic acids
disclosed herein. By "degenerate to" is meant that a different
three-letter codon is used to specify a particular amino acid.
[0192] As used herein, "epitope" refers to an antigenic determinant
of a polypeptide. An epitope could comprise 3 amino acids in a
spatial conformation which is unique to the epitope. Generally an
epitope consists of at least 5 such amino acids, and more usually,
consists of at least 8-10 such amino acids. Methods of determining
the spatial conformation of amino acids are known in the art, and
include, for example, x-ray crystallography and 2-dimensional
nuclear magnetic resonance.
[0193] As used herein, a "mimotope" is a peptide that mimics an
authentic antigenic epitope.
[0194] As used herein, the term "coat protein(s)" refers to the
protein(s) of a bacteriophage or a RNA-phage capable of being
incorporated within the capsid assembly of the bacteriophage or the
RNA-phage.
[0195] As used herein, a "coat polypeptide" as defined herein is a
polypeptide fragment of the coat protein that possesses coat
protein function and additionally encompasses the full length coat
protein as well or single-chain variants thereof.
[0196] A nucleic acid molecule is "operatively linked" to, or
"operably associated with", an expression control sequence when the
expression control sequence controls and regulates the
transcription and translation of nucleic acid sequence. The term
"operatively linked" includes having an appropriate start signal
(e.g., ATG) in front of the nucleic acid sequence to be expressed
and maintaining the correct reading frame to permit expression of
the nucleic acid sequence under the control of the expression
control sequence and production of the desired product encoded by
the nucleic acid sequence. If a gene that one desires to insert
into a recombinant DNA molecule does not contain an appropriate
start signal, such a start signal can be inserted in front of the
gene.
[0197] The term "stringent hybridization conditions" are known to
those skilled in the art and can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
A preferred, non-limiting example of stringent hybridization
conditions is hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2..times.SSC, 0.1% SDS at 50.degree. C., preferably at
55.degree. C., and more preferably at 60.degree. C. or 65.degree.
C.
Production of Virus-Like Particles
[0198] A more detailed description of the production of viral-like
particles is presented hereinafter (e.g. Example 1). The general
principles applicable to the production of viral-like particles can
be summarized as follows.
[0199] Phage display is one of several technologies that make
possible the presentation of large libraries of random amino acid
sequences with the purpose of selecting from them peptides with
certain specific functions. The basic idea is to create recombinant
bacteriophage genomes containing a library of randomized sequences
genetically fused to one of the structural proteins of the
virion.
[0200] When such recombinants are transfected into bacteria each
produces virus particles that display a particular peptide on their
surface and which package the same recombinant genome that encodes
that peptide, thus establishing the linkage of genotype and
phenotype essential to the method. Arbitrary functions (e.g. the
binding of a receptor, immunogenicity) can be selected from such
libraries by the use of biopanning and other techniques. Because of
constraints imposed by the need to transform and subsequently
cultivate bacteria, the practical upper limit on peptide library
complexity in phage display is said to be around
10.sup.10-10.sup.11 [Smothers et al., 2002, Science 298:621-622].
This requirement for passage through E. coli is the result of the
relatively complex makeup of the virions of the phages used for
phage display, and the consequent necessity that their components
be synthesized and assembled in vivo. For example, display of
certain peptides is restricted when filamentous phage is used, or
not possible, since the fused peptide has to be secreted through
the E. coli membranes as part of the phage assembly apparatus.
Bacteriophages
[0201] Properties of single-strand RNA bacteriophages are
disclosed, e.g. in The Bacteriophages, Calendar, R L, ed. Oxford
University Press. 2005. The known viruses of this group attack
bacteria as diverse as E. coli, Pseudomonas and Acinetobacter. Each
possesses a highly similar genome organization, replication
strategy, and virion structure. In particular, the bacteriophages
contain a single-stranded (+)-sense RNA genome, contain maturase,
coat and replicase genes, and have small (<300 angstrom)
icosahedral capsids. These include but are not limited to MS2, Qb,
R17, SP, PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNA
bacteriophages.
[0202] For purposes of illustration, the genome of a particularly
well-characterized member of the group, called MS2, comprises a
single strand of (+)-sense RNA 3569 nucleotides long, encoding only
four proteins, two of which are structural components of the
virion. The viral particle is comprised of an icosahedral capsid
made of 180 copies of coat protein and one molecule of maturase
protein together with one molecule of the RNA genome. Coat protein
is also a specific RNA binding protein. Assembly may possibly be
initiated when coat protein associates with its specific
recognition target an RNA hairpin near the 5'-end of the replicase
cistron. The virus particle is then liberated into the medium when
the cell bursts under the influence of the viral lysis protein. The
formation of an infectious virus requires at least three
components, namely coat protein, maturase and viral genome RNA, but
experiments show that the information required for assembly of the
icosahedral capsid shell is contained entirely within coat protein
itself. For example, purified coat protein can form capsids in
vitro in a process stimulated by the presence of RNA [Beckett et
al., 1988, J. Mol Biol 204: 939-47]. Moreover, coat protein
expressed in cells from a plasmid assembles into a virus-like
particle in vivo [Peabody, D. S., 1990, J Biol Chem 265:
5684-5689].
Coat Polypeptide
[0203] The coat polypeptide encoded by the coding region is
typically at least 120, preferably, at least 125 amino acids in
length, and no greater than 135 amino acids in length, preferably,
no greater than 130 amino acids in length. It is expected that a
coat polypeptide from essentially any single-stranded RNA
bacteriophage can be used. Examples of coat polypeptides include
but are not limited to the MS2 coat polypeptide, R17 coat
polypeptide (see, for example, Genbank Accession No P03612), PRR1
coat polypeptide (see, for example, Genbank Accesssion No.
ABH03627), fr phage coat polypeptide (see, for example, Genbank
Accession No. NP.sub.--039624), GA coat polypeptide (see, for
example, Genbank Accession No. P07234), Qb coat polypeptide (see,
for example, Genbank Accession No. P03615), SP coat polypeptide
(see, for example, Genbank Accession No P09673), f4 coat
polypeptide (see, for example, Genbank accession no. M37979.1 and
PP7 coat polypeptide (see, for example, Genbank Accession No PO363
0).
[0204] Examples of PP7 coat polypeptides include but are not
limited to the various chains of PP7 Coat Protein Dimer in Complex
With Rna Hairpin (e.g. Genbank Accession Nos. 2QUXR; 2QUXO; 2QUX_L;
2QUX_I; 2QUX_F; and 2QUX_C). See also Example 1 herein and Peabody,
et al., RNA recognition site of PP7 coat protein, Nucleic Acids
Research, 2002, Vol. 30, No. 19 4138-4144.
[0205] The coat polypeptides useful in the present invention also
include those having similarity with one or more of the coat
polypeptide sequences disclosed above. The similarity is referred
to as structural similarity. Structural similarity may be
determined by aligning the residues of the two amino acid sequences
(i.e., a candidate amino acid sequence and the amino acid sequence)
to optimize the number of identical amino acids along the lengths
of their sequences; gaps in either or both sequences are permitted
in making the alignment in order to optimize the number of
identical amino acids, although the amino acids in each sequence
must nonetheless remain in their proper order. A candidate amino
acid sequence can be isolated from a single stranded RNA virus, or
can be produced using recombinant techniques, or chemically or
enzymatically synthesized. Preferably, two amino acid sequences are
compared using the BESTFIT algorithm in the GCG package (version
10.2, Madison Wis.), or the Blastp program of the BLAST 2 search
algorithm, as described by Tatusova, et al. (FEMS Microbial Lett
1999, 174:247-250), and available at
http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html. Preferably, the
default values for all BLAST 2 search parameters are used,
including matrix=BLOSUM62; open gap penalty=11, extension gap
penalty=1, gap xdropoff=50, expect=10, wordsize=3, and optionally,
filter on. In the comparison of two amino acid sequences using the
BLAST search algorithm, structural similarity is referred to as
"identities."
[0206] Preferably, a coat polypeptide also includes polypeptides
with an amino acid sequence having at least 80% amino acid
identity, at least 85% amino acid identity, at least 90% amino acid
identity, or at least 95% amino acid identity to one or more of the
amino acid sequences disclosed above. Preferably, a coat
polypeptide is active. Whether a coat polypeptide is active can be
determined by evaluating the ability of the polypeptide to form a
capsid and package a single stranded RNA molecule. Such an
evaluation can be done using an in vivo or in vitro system, and
such methods are known in the art and routine. Alternatively, a
polypeptide may be considered to be structurally similar if it has
similar three dimensional structure as the recited coat polypeptide
and/or functional activity.
[0207] Heterologous peptide sequences (in the present invention, at
least one CRLF-2 peptide as otherwise disclosed herein) inserted
into the coat polypeptide or polypeptide may be a random peptide
sequence. In a particular embodiment, the random sequence has the
sequence Xaa.sub.n wherein n is at least 4, at least 6, or at least
8 and no greater than 20, no greater than 18, or no greater than
16, and each Xaa is independently a random amino acid.
Alternatively, the peptide fragment may possess a known
functionality (e.g., antigenicity, immunogenicity). The
heterologous sequence may be present at the amino-terminal end of a
coat polypeptide, at the carboxy-terminal end of a coat
polypeptide, or present elsewhere within the coat polypeptide,
preferably in the AB loop. Preferably, the heterologous sequence is
present at a location in the coat polypeptide such that the
inserted sequence is expressed on the outer surface of the capsid.
In a particular embodiment, the peptide sequence may be inserted
into the AB loop regions the above-mentioned coat polypeptides.
Examples of such locations include, for instance, insertion of the
heterologous peptide sequence into a coat polypeptide immediately
following amino acids 11-17, or amino acids 13-17 of the coat
polypeptide. In a most particular embodiment, the heterologous
peptide is inserted at a site corresponding to amino acids 11-17 or
particularly 13-17 of MS-2.
[0208] Alternatively, the heterologous peptide may be inserted at
the N-terminus or C-terminus of the coat polypeptide. Any one or
more of the CRLF-2 peptides as described herein may be used as the
heterologous peptides for insertion into the coat polypeptide which
produces a VLP expressing the inserted CRLF-2 peptide on its
surface.
[0209] The heterologous peptide may be selected from the group
consisting of a peptide that targets CRLF-2 and/or CD 19, a
receptor for CRLF-2 and/or CD 19, a ligand which binds to a CRLF-2
and/or CD 19 cell surface receptor, a peptide with affinity for
either end of a filamentous phage particle specific to CRLF-2
and/or CD 19, a metal binding peptide that binds to CRLF-2 and/or
CD 19 or a CRLF-2 and/or CD 19 peptide with affinity for the
surface of MS2. In preferred aspects, the heterologous peptide
consists essentially of or is specifically MTAAPVH (SEQ ID NO: 4),
LTTPNWV (SEQ ID NO:5), AAQTSTP (SEQ ID NO:6), TDAHASV (SEQ ID
NO:7), FSYLPSH (SEQ ID NO: 8), YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ
ID NO:10), AATLFPL (SEQ ID NO:11), LTSRPTL (SEQ ID NO: 12), ETKAWWL
(SEQ ID NO:13), HWGMWSY (SEQ ID NO:14), SQIFGNK (SEQ ID NO:15),
SQAFVLV (SEQ ID NO:16), WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID
NO:18), TMCIYCT (SEQ ID NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW
(SEQ ID NO:21) and NILSLSM (SEQ ID NO:22). Preferred CRLF-2 binding
peptides include MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5),
AAQTSTP (SEQ ID NO:6), MHAPPFY (SEQ ID NO:10), ETKAWWL (SEQ ID
NO:13), SQIFGNK (SEQ ID NO:15), AATLFPL (SEQ ID NO:11), TDAHASV
(SEQ ID NO:7) and FSYLPSH (SEQ ID NO: 8). More preferably, the
CRLF-2 binding peptide is MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID
NO:5), AAQTSTP (SEQ ID NO:6) or MHAPPFY (SEQ ID NO:10). Often, the
CRLF-2 binding peptide used in embodiments according to the present
invention includes MTAAPVH (SEQ ID NO: 4) and LTTPNWV (SEQ ID
NO:5). Most often, the CRLF-2 binding peptide is MTAAPVH (SEQ ID
NO: 4).
[0210] In order to determine a corresponding position in a
structurally similar coat polypeptide, the amino acid sequence of
this structurally similar coat polypeptide is aligned with the
sequence of the named coat polypeptide as specified above.
[0211] In a particular embodiment, the coat polypeptide is a
single-chain dimer containing an upstream and downstream subunit.
Each subunit contains a functional coat polypeptide sequence. The
heterologous peptide may be inserted ton the upstream and/or
downstream subunit at the sites mentioned herein above, e.g., A-B
loop region of downstream subunit. In a particular embodiment, the
coat polypeptide is a single chain dimer of an MS2 or PP7 coat
polypeptide.
Preparation of Transcription Unit
[0212] The transcription unit of the present invention comprises an
expression regulatory region, (e.g., a promoter), a sequence
encoding a coat polypeptide and transcription terminator. The RNA
polynucleotide may optionally include a coat recognition site (also
referred to a "packaging signal", "translational operator
sequence", "coat recognition site"). Alternatively, the
transcription unit may be free of the translational operator
sequence.
[0213] The promoter, coding region, transcription terminator, and,
when present, the coat recognition site, are generally operably
linked. "Operably linked" or "operably associated with" refer to a
juxtaposition wherein the components so described are in a
relationship permitting them to function in their intended manner.
A regulatory sequence is "operably linked" to, or "operably
associated with", a coding region when it is joined in such a way
that expression of the coding region is achieved under conditions
compatible with the regulatory sequence. The coat recognition site,
when present, may be at any location within the RNA polynucleotide
provided it functions in the intended manner.
[0214] The invention is not limited by the use of any particular
promoter, and a wide variety of promoters are known. The promoter
used in the invention can be a constitutive or an inducible
promoter. Preferred promoters are able to drive high levels of RNA
encoded by me coding region encoding the coat polypeptide Examples
of such promoters are known in the art and include, for instance,
T7, T3, and SP6 promoters.
[0215] The nucleotide sequences of the coding regions encoding coat
polypeptides described herein are readily determined. These classes
of nucleotide sequences are large but finite, and the nucleotide
sequence of each member of the class can be readily determined by
one skilled in the art by reference to the standard genetic
code.
[0216] Furthermore, the coding sequence of an RNA bacteriophage
single chain coat polypeptide comprises a site for insertion of a
heterologous peptide as well as a coding sequence for the
heterologous peptide itself. In a particular embodiment, the site
for insertion of the heterologous peptide is a restriction enzyme
site.
[0217] In a particular embodiment, the coding region encodes a
single-chain dimer of the coat polypeptide. In a most particular
embodiment, the coding region encodes a modified single chain coat
polypeptide dimer, where the modification comprises an insertion of
a coding sequence at least four amino acids at the insertion site.
The transcription unit may contain a bacterial promoter, such as a
lac promoter or it may contain a bacteriophage promoter, such as a
T7 promoter and optionally a T7 transcription terminator.
[0218] In addition to containing a promoter and a coding region
encoding a fusion polypeptide, the RNA polynucleotide typically
includes a transcription terminator, and optionally, a coat
recognition site. A coat recognition site is a nucleotide sequence
that forms a hairpin when present as RNA. This is also referred to
in the art as a translational operator, a packaging signal, and an
RNA binding site. Without intending to be limiting, this structure
is believed to act as the binding site recognized by the
translational repressor (e.g., the coat polypeptide), and initiate
RNA packaging. The nucleotide sequences of coat recognition sites
are known in the art. Other coat recognition sequences have been
characterized in the single stranded RNA bacteriophages R17, GA,
Q.beta., SP, and PP7, and are readily available to the skilled
person. Essentially any transcriptional terminator can be used in
the RNA polynucleotide, provided it functions with the promoter.
Transcriptional terminators are known to the skilled person,
readily available, and routinely used.
Synthesis
[0219] The VLPs of the present invention may be synthesized in
vitro in a coupled cell-free transcription/translation system.
Alternatively VLPs could be produced in vivo by introducing
transcription units into bacteria, especially if transcription
units contain a bacterial promoter.
Assembly of VLPs Encapsidating Heterologous Substances
[0220] As noted above, the VLPs of the present invention may
encapsidate one or more peptides that target CRLF-2 and/or CD19.
These VLPs play be assembled by performing an in vitro VLP assembly
reaction in the presence of the heterologous substance.
Specifically, purified coat protein subunits are obtained from VLPs
that have been disaggregated with a denaturant (usually acetic
acid). The protein subunits are mixed with the heterologous
substance. In a particular embodiment, the substance has some
affinity for the interior of the VLP and is preferably negatively
charged.
[0221] Another method involves attaching the heterologous substance
to a synthetic RNA version of the translational operator. During an
in vitro assembly reaction the RNA will tightly bind to its
recognition site and be efficiently incorporated into the resulting
VLP, carrying with it the foreign substance.
[0222] In another embodiment, the substance is passively diffused
into the VLP through pores that naturally exist in the VLP surface.
In a particular embodiment, the substance is small enough to pass
through these pores (in MS2 they are about 10 angstroms diameter)
and has a high affinity for the interior of the VLP.
VLP Populations
[0223] In the term "VLP populations or libraries", "population" and
"libraries" are used interchangeably and are thus deemed to be
synonymous. In one particular embodiment, the library may be a
random library; in another embodiment, the library is an CRLF-2
and/or CD 19-targeting peptide fragment library or a library of
CRLF-2 and/or CD 19-targeting peptide fragments derived from CRLF-2
and/or CD19 polypeptides.
Random Libraries (Populations)
[0224] Oligonucleotides encoding peptides may be prepared. In one
particular embodiment, the triplets encoding a particular amino
acid have the composition NNS where N is A, G, C or T and S is G or
T or alternatively NNY where N is A, G, C, or T and Y is C or T. In
order to minimize the presence of stop codons, peptide libraries
can be constructed using oligonucleotides synthesized from custom
trinucleotide phosphoramidite mixtures (available from Glen
Research, Inc.) designed to more accurately reflect natural amino
acid compositions and completely lacking stop codons.
Antigen Fragment Libraries
[0225] An alternative strategy takes advantage of the existence of
a cloned gene or genome to create random fragment libraries. The
idea is to randomly fragment the gene (e.g. with DNasel) to an
appropriate average size (e.g. -30 bp), and to blunt-end ligate the
fragments to an appropriate site in coat polypeptide. In a
particular embodiment, a restriction site may be inserted into the
AB-loop or N-terminus of the coat polypeptide). Only a minority of
clones will carry productive inserts, because they shift reading
frame, introduce a stop codon, or receive an insert in antisense
orientation, Any expression vector may in one embodiment contain a
marker to pre-select clones with intact coat coding sequences. For
example, GalE-strains of E. coli are defective for galactose kinase
and accumulate a toxic metabolite when b-galactosidase is expressed
in the presence of the galactose analogue, phenyl-b,D-galactoside
(PGaI). Subjecting a random antigen-fragment library to selection
for translational repressor function in the GalE-strain CSH41
F-containing pRZ5, a plasmid that fuses the MS2 replicase cistron's
translational operator to lacZ will eliminate most undesired
insertions by enriching the library for those that at least
maintain the coat reading-frame.
Synthesis
[0226] In a particular embodiment, VLP populations may be
synthesized in a coupled in vitro transcription/translation system
using procedures known in the art (see, for example, U.S. Pat. No.
7,008,651 Kramer et al., 1999, Cell-free coupled
transcription-translation systems from E. coli, In. Protein
Expression. A Practical Approach, Higgins and Hames (eds.), Oxford
University Press). In a particular embodiment, bacteriophage T7 (or
a related) RNA polymerase is used to direct the high-level
transcription of genes cloned under control of a T7 promoter in
systems optimized to efficiently translate the large amounts of RNA
thus produced [for examples, see Kim et al., 1996, Eur J Biochem
239: 88 1-886; Jewett et al., 2004, Biotech and Bioeng 86:
19-26].
[0227] It is possible in a mixture of templates, particularly in
the population of the present invention, different individual coat
polypeptides, distinguished by their fusion to different peptides,
could presumably package each other's mRNAs, thus destroying the
genotype/phenotype linkage needed for effective phage display.
Moreover, because each capsid is assembled from multiple subunits,
formation of hybrid capsids may occur. Thus, in one preferred
embodiment, when preparing the populations or libraries of the
present invention, one or more cycles of the
transcription/translation reactions be performed in water/oil
emulsions (Tawfik et al., 1998, Nat Biotechnol 16: 652-6). In this
now well-established method, individual templates are segregated
into the aqueous compartments of a water/oil emulsion. Under
appropriate conditions huge numbers of aqueous microdroplets can be
formed, each containing on average a single DNA template molecule
and the machinery of transcription/translation. Because they are
surrounded by oil, these compartments do not communicate with one
another. The coat polypeptides synthesized in such droplets should
associate specifically with the same mRNAs which encode them, and
ought to assemble into capsids displaying only one peptide. After
synthesis, the emulsion can be broken and the capsids recovered and
subjected to selection. In one particular embodiment, all of the
transcription/translation reactions are performed in the water/oil
emulsion. In one particular embodiment, only droplets containing
only one template per droplet (capsids displaying only one peptide)
is isolated. In another embodiment, droplets containing mixed
capsids may be isolated (plurality of templates per droplet) in one
or more cycles of transcription/translation reactions and
subsequently capsids displaying only one peptide (one template per
droplet) are isolated.
Selection of CRLF-2 and/or CD19-Targeting Candidates
[0228] The VLP populations or libraries of the present invention
may be used to select CRLF-2 and/or CD19-targeting candidates. The
libraries may be random or antigenic libraries. Libraries of random
or alternatively antigen-derived peptide sequences are displayed on
the surface of VLPs, and specific target epitopes, or perhaps
mimotopes are then isolated by affinity-selection using antibodies.
Since the VLPs encapsidate their own mRNAs, sequences encoding them
(and their guest peptides) can be recovered by reverse
transcription and PCR. Individual affinity-selected VLPs are
subsequently cloned, over-expressed and purified.
[0229] Techniques for affinity selection in phage display are well
developed and are directly applicable to the VLP display system of
the present invention. Briefly, an antibody (or antiserum) is
allowed to form complexes with the peptides on VLPs in a random
sequence or antigen fragment display library. Typically the
antibodies will have been labeled with biotin so that the complexes
can be captured by binding to a streptavidin-coated surface,
magnetic beads, or other suitable immobilizing medium.
[0230] After washing, bound VLPs are eluted, and RNAs are extracted
from the affinity-selected population and subjected to reverse
transcription and PCR to recover the coat-encoding sequences, which
are then recloned and subjected to further rounds of expression and
affinity selection until the best-binding variants are obtained. A
number of schemes for retrieval of RNA from VLPs are readily
imagined. One attractive possibility is to simply capture
biotin-mAb-VLP complexes in streptavidin coated PCR tubes, then
thermally denature the VLPs and subject their RNA contents directly
to RT-PCR. Many obvious alternatives exist and adjustments may be
required depending on considerations such as the binding capacities
of the various immobilizing media. Once the selected sequences are
recovered by RT-PCR it is a simple matter to clone and reintroduce
them into E coli, taking care at each stage to preserve the
requisite library diversity, which, of course, diminishes with each
round of selection. When selection is complete, each clone can be
over-expressed to produce a CRLF-2 and/or CD19-targeting VLP.
[0231] VLPs according to the present invention, as described above,
express at least one CRLF-2 peptide on the surface of the VLP and
preferably, include cargo, for example at least one anticancer drug
(especially, for example, a chemotherapeutic agent as otherwise
described herein which and optionally additional cargo such as one
or more of a fusogenic peptide that promotes endosomal escape of
VLPs and encapsulated DNA, other cargo comprising at least one
cargo component selected from the group consisting of double
stranded linear DNA or a plasmid DNA, an imaging agent, small
interfering RNA, small hairpin RNA, microRNA, or a mixture thereof,
wherein one of said cargo components is optionally conjugated
further with a nuclear localization sequence. Any one or more of
these components may be incorporated into VLPs readily using
methods well-known in the art, including modifying the pac site of
the bacteriophage RNA using crosslinking agents and conjugating the
various components onto the crosslinking agents within the dimer
coat polypeptide without impacting the ability of the coat
polypeptide to spontaneously reassemble into VLPs as described in
U.S. patent application Ser. No. 12/960,168, filed Dec. 3, 2010,
entitled "Virus-Like Particles as Targeted Multifunctional
Nanocarriers for Delivery of Drugs, Therapeutics, Sensors and
Contrast Agents to Arbitrary Cell Types", which is incorporated by
reference in its entirety herein.
[0232] Bacteriophage VLPs such as MS2 and/or Q.beta.
bacteriophages, also self-assemble into complete capsides in the
presence of nucleic acids and thus, can be used to specifically
encapsidate therapeutic RNA (e.g., shRNA, siRNA, antisense
oligonucleotides, other microRNAs, ribozymes, RNA decoys, aptamers)
and other RNA-modified cargos, including one or more RNA-modified
cytotoxic agents (e.g., chemotherapeutic drugs or toxins) or one or
more RNA-modified imaging agents (e.g. quantum dots). Typically,
the nucleic acid is conjugated to one or more cytotoxic agents or
one or more imaging agents using an appropriate crosslinking
molecule as described herein.
[0233] For example, a chemotherapeutic agent, such as doxorubicin,
can be conjugated to the pac site of MS2 using a heterobifunctional
crosslinker molecule (e.g., NHA ester-maleimide agent, among
others) to link a primary amine moiety present in doxorubicin or
other chemotherapeutic agent to a nucleic acid molecule including,
for example, the pac site) that is modified with a 3' or 5'
sulfhydryl group. In exemplary approaches, cargo components,
including, for example, drugs, therapeutic RNA as otherwise
described herein, quantum dots, gold nanoparticles, iron oxide
nanoparticles, etc. and other cargo, etc. can be linked to the
thiolated pac site and incorporated with the capsids of the VLPs.
Approaches for incorporating the various components into VLPs
(preferably by conjugation through a crosslinking agent at the pac
site) pursuant to the present invention are well-known in the
art.
[0234] The efficacy and rate of capside assembly are maximized in
the presence of the MS2 translational operator, a 19-nucleotide RNA
stem-loop (SEQ ID NO:32, SEQ ID NO:33), that via its interaction
with coat protein, mediates exclusive encapsidation of the MS2
genome during bacteriophage replication. See, Wu, et al.,
Bioconjugate Chemistry, 6(5):587-595 (1995); Pickett & Peabody,
Nucl Acids. Res., 21 (19):4621-4626 (1993) and Uhlenback, Nature
Structure Biology, 5(3):174-174 (1998). The MS2 operator, or pac
site, can promote efficient encapsidation of non-genomic materials,
such as the polypeptide toxins, including the A-chain of ricin
toxin, among others, within the interior volume of MS2 VLPs upon
conjugation of the pac site to the cargo of interest. MS2 VLPs will
also encapsidate RNA hairpins with sequences that differ from that
of the native operator, as well as heterologous nucleic acids,
including singe- and double-stranded RNA and DNA less than 3 bkp in
length. Accordingly, the sequence of the pac site can be modified
as long as the modification does not prevent the RNA molecule from
inducing VLP self assembly. For example, the pac site can further
comprise a spacer molecule, such as a polyU nucleotide (e.g.
(U).sub.3-9)).
[0235] Using known methods, a polyethylene glycol moiety may be
attached to the VLPs or protocells as otherwise described herein.
PEGylation sometimes assists in minimizing proteolytic degradation,
reducing the humoral immune response against the capside protein
and reducing non-specific interactions with non-target cells and
thus, can help to increase the circulation half-life and enhance
the bioavailability of the encapsidated cargo without appreciably
affecting the specific affinity of the nanoparticle for their
target cells.
[0236] The term "reporter" is used to describe an imaging agent or
moiety which is incorporated into the phospholipid bilayer or cargo
of protocells according to an embodiment of the present invention
and provides a signal which can be measured. The moiety may provide
a fluorescent signal or may be a radioisotope which allows
radiation detection, among others. Exemplary fluorescent labels for
use in protocells (preferably via conjugation or adsorption to the
lipid bilayer or silica core, although these labels may also be
incorporated into cargo elements such as DNA, RNA, polypeptides and
small molecules which are delivered to cells by the protocells,
include Hoechst 33342 (350/461), 4',6-diamidino-2-phenylindole
(DAPI, 356/451), Alexa Fluor.RTM. 405 carboxylic acid, succinimidyl
ester (401/421), CellTracker.TM. Violet BMQC (415/516),
CellTracker.TM. Green CMFDA (492/517), calcein (495/515), Alexa
Fluor.RTM. 488 conjugate of annexin V (495/519), Alexa Fluor.RTM.
488 goat anti-mouse IgG (H+L) (495/519), Click-iT.RTM. AHA Alexa
Fluor.RTM. 488 Protein Synthesis HCS Assay (495/519),
LIVE/DEAD.RTM. Fixable Green Dead Cell Stain Kit (495/519),
SYTOX.RTM. Green nucleic acid stain (504/523), MitoSOX.TM. Red
mitochondrial superoxide indicator (510/580). Alexa Fluor.RTM. 532
carboxylic acid, succinimidyl ester (532/554), pHrodo.TM.
succinimidyl ester (558/576), CellTracker.TM. Red CMTPX (577/602),
Texas Red.RTM. 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine
(Texas Red.RTM. DHPE, 583/608), Alexa Fluor.RTM. 647 hydrazide
(649/666), Alexa Fluor.RTM. 647 carboxylic acid, succinimidyl ester
(650/668), Ulysis.TM. Alexa Fluor.RTM. 647 Nucleic Acid Labeling
Kit (650/670) and Alexa Fluor.RTM. 647 conjugate of annexin V
(650/665). Moities which enhance the fluorescent signal or slow the
fluorescent fading may also be incorporated and include
SlowFade.RTM. Gold antifade reagent (with and without DAPI) and
Image-iT.RTM. FX signal enhancer. All of these are well known in
the art. Additional reporters include polypeptide reporters which
may be expressed by plasmids (such as histone-packaged supercoiled
DNA plasmids) and include polypeptide reporters such as fluorescent
green protein and fluorescent red protein. Reporters pursuant to
the present invention are utilized principally in diagnostic
applications including diagnosing the existence or progression of
cancer (cancer tissue) in a patient and or the progress of therapy
in a patient or subject.
[0237] The term "histone-packaged supercoiled plasmid DNA" is used
to describe a preferred component of protocells according to the
present invention which utilize a preferred plasmid DNA which has
been "supercoiled" (i.e., folded in on itself using a
supersaturated salt solution or other ionic solution which causes
the plasmid to fold in on itself and "supercoil" in order to become
more dense for efficient packaging into the protocells). The
plasmid may be virtually any plasmid which expresses any number of
polypeptides or encode RNA, including small hairpin RNA/shRNA or
small interfering RNA/siRNA, as otherwise described herein. Once
supercoiled (using the concentrated salt or other anionic
solution), the supercoiled plasmid DNA is then complexed with
histone proteins to produce a histone-packaged "complexed"
supercoiled plasmid DNA.
[0238] "Packaged" DNA herein refers to DNA that is loaded into
protocells (either adsorbed into the pores or confined directly
within the nanoporous silica core itself). To minimize the DNA
spatially, it is often packaged, which can be accomplished in
several different ways, from adjusting the charge of the
surrounding medium to creation of small complexes of the DNA with,
for example, lipids, proteins, or other nanoparticles (usually,
although not exclusively cationic). Packaged DNA is often achieved
via lipoplexes (i.e. complexing DNA with cationic lipid mixtures).
In addition, DNA has also been packaged with cationic proteins
(including proteins other than histones), as well as gold
nanoparticles (e.g. NanoFlares--an engineered DNA and metal complex
in which the core of the nanoparticle is gold).
[0239] Any number of histone proteins, as well as other means to
package the DNA into a smaller volume such as normally cationic
nanoparticles, lipids, or proteins, may be used to package the
supercoiled plasmid DNA "histone-packaged supercoiled plasmid DNA",
but in therapeutic aspects which relate to treating human patients,
the use of human histone proteins are preferably used. In certain
aspects of the invention, a combination of human histone proteins
H1, H2A, H2B, H3 and H4 in a preferred ratio of 1:2:2:2:2, although
other histone proteins may be used in other, similar ratios, as is
known in the art or may be readily practiced pursuant to the
teachings of the present invention. The DNA may also be double
stranded linear DNA, instead of plasmid DNA, which also may be
optionally supercoiled and/or packaged with histones or other
packaging components.
[0240] Other histone proteins which may be used in this aspect of
the invention include, for example, H1F, H1F0, H1FNT, H1FOO, H1FX
H1H1 HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E, HIST1H1T;
H2AF, H2AFB1, H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2,
H2AFZ, H2A1, HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE,
HIST1H2AG, HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL, HIST1H2AM,
H2A2, HIST2H2AA3, HIST2H2AC, H2BF, H2BFM, HSBFS, HSBFWT, H2B1,
HIST1H2BA, HIST1HSBB, HIST1HSBC, HIST1HSBD, HIST1H2BE, HIST1H2BF,
HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK, HIST1H2BL,
HIST1H2BM, HIST1H2BN, HIST1H2BO, H2B2, HIST2H2BE, H3A1, HIST1H3A,
HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G,
HIST1H3H, HIST1H3I, HIST1H3J, H3A2, HIST2H3C, H3A3, HIST3H3, H41,
HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F,
HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, H44 and
HIST4H4.
[0241] The term "nuclear localization sequence" refers to a peptide
sequence incorporated or otherwise crosslinked into histone
proteins which comprise the histone-packaged supercoiled plasmid
DNA. In certain embodiments, protocells according to the present
invention may further comprise a plasmid (often a histone-packaged
supercoiled plasmid DNA) which is modified (crosslinked) with a
nuclear localization sequence (note that the histone proteins may
be crosslinked with the nuclear localization sequence or the
plasmid itself can be modified to express a nuclear localization
sequence) which enhances the ability of the histone-packaged
plasmid to penetrate the nucleus of a cell and deposit its contents
there (to facilitate expression and ultimately cell death. These
peptide sequences assist in carrying the histone-packaged plasmid
DNA and the associated histones into the nucleus of a targeted cell
whereupon the plasmid will express peptides and/or nucleotides as
desired to deliver therapeutic and/or diagnostic molecules
(polypeptide and/or nucleotide) into the nucleus of the targeted
cell. Any number of crosslinking agents, well known in the art, may
be used to covalently link a nuclear localization sequence to a
histone protein (often at a lysine group or other group which has a
nucleophilic or electrophilic group in the side chain of the amino
acid exposed pendant to the polypeptide) which can be used to
introduce the histone packaged plasmid into the nucleus of a cell.
Alternatively, a nucleotide sequence which expresses the nuclear
localization sequence can be positioned in a plasmid in proximity
to that which expresses histone protein such that the expression of
the histone protein conjugated to the nuclear localization sequence
will occur thus facilitating transfer of a plasmid into the nucleus
of a targeted cell.
[0242] Proteins gain entry into the nucleus through the nuclear
envelope. The nuclear envelope consists of concentric membranes,
the outer and the inner membrane. These are the gateways to the
nucleus. The envelope consists of pores or large nuclear complexes.
A protein translated with a NLS will bind strongly to importin (aka
karyopherin), and together, the complex will move through the
nuclear pore. Any number of nuclear localization sequences may be
used to introduce histone-packaged plasmid DNA into the nucleus of
a cell. Preferred nuclear localization sequences include
GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY SEQ I.D NO: 28, RRMKWKK
(SEQ ID NO:29), PKKKRKV (SEQ ID NO: 30), and KR[PAATKKAGQA]KKKK
(SEQ ID NO:31), the NLS of nucleoplasmin, a prototypical bipartite
signal comprising two clusters of basic amino acids, separated by a
spacer of about 10 amino acids. Numerous other nuclear localization
sequences are well known in the art. See, for example, LaCasse, et
al., Nuclear localization signals overlap DNA- or RNA-binding
domains in nucleic acid-binding proteins. Nucl. Acids Res., 23,
1647-16561995); Weis, K. Importins and exportins: how to get in and
out of the nucleus [published erratum appears in Trends Biochem Sci
1998 July; 23(7):235]. TIBS, 23, 185-9 (1998); and Murat Cokol, Raj
Nair & Burkhard Rost, "Finding nuclear localization signals",
at the website ubic.bioc.columbia.edu/papers/2000
nls/paper.html#tab2.
[0243] "Tyrosine kinase inhibitors" include, but are not limited to
imatinib, axitinib, bosutinib, cediranib, dasatinib, erlotinib,
gefitinib, lapatinib, lestaurtinib, nilotinib, semaxanib,
sunitinib, toceranib, vandetanib, vatalanib, sorafenib
(Nexavar.RTM.), lapatinib, motesanib, vandetanib (Zactima.RTM.),
MP-412, lestaurtinib, XL647, XL999, tandutinib, PKC412, AEE788,
OSI-930, OSI-817, sunitinib maleate (Sutent.RTM.)) and
N-(4-(4-aminothieno[2,3-d]pyrimidin-5-yl)phenyl)-N-(2-fluoro-5-(trifluor--
omethyl)phenyl)urea, the preparation of which is described in
United States Patent Application Document No. 2007/0155758.
[0244] The term "tyrosine kinase inhibitors" is intended to
encompass the hydrates, solvates (such as alcoholates), polymorphs,
N-oxides, and pharmaceutically acceptable acid or base addition
salts of tyrosine kinase inhibiting compounds.
[0245] The term "effective" is used herein, unless otherwise
indicated, to describe an amount of a compound or composition
which, in context, is used to produce or affect an intended result,
whether that result relates to treating a subject who suffers from
cancer and symptoms and conditions associated with cancer. This
term subsumes all other effective amount or effective concentration
terms which are otherwise described in the present application.
[0246] The term "inhibitory effective concentration" or "inhibitory
effective amount" describes concentrations or amounts of compounds
that, when administered according to the present invention,
substantially or significantly inhibit aspects or symptoms of
cancer or conditions associated with cancer.
[0247] The term "preventing effective amount" describes
concentrations or amounts of compounds which, when administered
according to the present invention, are prophylactically effective
in preventing or reducing the likelihood of the onset of cancer or
a condition associated with cancer or in ameliorating the symptoms
of such disorders or symptoms. The terms inhibitory effective
amount or preventive effective amount also generally fall under the
rubric "effective amount".
[0248] In certain embodiments, acute lymophblastic leukemia (ALL),
including B-precursor acute lymphoblastic leukemia (B-ALL) is
predicted to be either responsive or non-responsive to tyrosine
kinase inhibitor mono or co-therapy based on a determination of
whether it is likely to result in one or more of the clinical
outcomes outlined in the following excerpts from the National
Cancer Institute Childhood Acute Lymphoblastic Leukemia Treatment
(PDQ.RTM.)
(http://www.cancer.gov/cancertopics/pdq/treatment/childALL/HealthProfessi-
onal/Page2#Section.sub.--526). (These clinical assessments and
prognosis indicia are purely exemplary and are not limiting. Other
clinical analyses may be employed in the determination of whether
ALL, including B-precursor acute lymphoblastic leukemia (B-ALL)
will respond to tyrosine kinase inhibitor mono or co-therapy.)
[0249] The rapidity with which leukemia cells are eliminated
following onset of treatment and the level of residual disease at
the end of induction are associated with long-term outcome. Because
treatment response is influenced by the drug sensitivity of
leukemic cells and host pharmacodynamics and pharmacogenomics,
early response has strong prognostic significance. Various ways of
evaluating the leukemia cell response to treatment have been
utilized, including the following:
[0250] 1. MRD determination.
[0251] 2. Day 7 and day 14 bone marrow responses.
[0252] 3. Peripheral blood response to steroid prophase.
[0253] 4. Peripheral blood response to multiagent induction
therapy.
[0254] 5. Induction failure.
MRD Determination.
[0255] Morphologic assessment of residual leukemia in blood or bone
marrow is often difficult and is relatively insensitive.
Traditionally, a cutoff of 5% blasts in the bone marrow (detected
by light microscopy) has been used to determine remission status.
This corresponds to a level of 1 in 20 malignant cells. If one
wishes to detect lower levels of leukemic cells in either blood or
marrow, specialized techniques such as PCR assays, which determine
unique Ig/T-cell receptor gene rearrangements, fusion transcripts
produced by chromosome translocations, or flow cytometric assays,
which detect leukemia-specific immunophenotypes, are required. With
these techniques, detection of as few as 1 leukemia cell in 100,000
normal cells is possible, and MRD at the level of 1 in 10,000 cells
can be detected routinely.
[0256] Multiple studies have demonstrated that end-induction MRD is
an important, independent predictor of outcome in children and
adolescents with B-lineage ALL. MRD response discriminates outcome
in subsets of patients defined by age, leukocyte count, and
cytogenetic abnormalities. Patients with higher levels of
end-induction MRD have a poorer prognosis than those with lower or
undetectable levels. End-induction MRD is used by almost all groups
as a factor determining the intensity of postinduction treatment,
with patients found to have higher levels allocated to more
intensive therapies. MRD levels at earlier (e.g., day 8 and day 15
of induction) and later time points (e.g., week 12 of therapy) also
predict outcome.
[0257] MRD measurements, in conjunction with other presenting
features, have also been used to identify subsets of patients with
an extremely low risk of relapse. The COG reported a very favorable
prognosis (5-year EFS of 97%.+-.1%) for patients with B-precursor
phenotype, NCI standard risk age/leukocyte count, CNS 1 status, and
favorable cytogenetic abnormalities (either high hyperdiploidy with
favorable trisomies or the ETV6-RUNX1 fusion) who had less than
0.01% MRD levels at both day 8 (from peripheral blood) and
end-induction (from bone marrow).
[0258] There are fewer studies documenting the prognostic
significance of MRD in T-cell ALL. In the AIEOP-BFM ALL 2000 trial,
MRD status at day 78 (week 12) was the most important predictor for
relapse in patients with T-cell ALL. Patients with detectable MRD
at end-induction who had negative MRD by day 78 did just as well as
patients who achieved MRD-negativity at the earlier end-induction
time point. Thus, unlike in B-cell precursor ALL, end-induction MRD
levels were irrelevant in those patients whose MRD was negative at
day 78. A high MRD level at day 78 was associated with a
significantly higher risk of relapse.
[0259] There are few studies of MRD in the CSF. In one study, MRD
was documented in about one-half of children at diagnosis. In this
study, CSF MRD was not found to be prognostic when intensive
chemotherapy was given.
[0260] Although MRD is the most important prognostic factor in
determining outcome, there are no data to conclusively show that
modifying therapy based on MRD determination significantly improves
outcome in newly diagnosed ALL.
Day 7 and Day 14 Bone Marrow Responses.
[0261] Patients who have a rapid reduction in leukemia cells to
less than 5% in their bone marrow within 7 or 14 days following
initiation of multiagent chemotherapy have a more favorable
prognosis than do patients who have slower clearance of leukemia
cells from the bone marrow.
Peripheral Blood Response to Steroid Prophase.
[0262] Patients with a reduction in peripheral blast count to less
than 1,000/4 after a 7-day induction prophase with prednisone and
one dose of intrathecal methotrexate (a good prednisone response)
have a more favorable prognosis than do patients whose peripheral
blast counts remain above 1,000/.mu.L (a poor prednisone response).
Poor prednisone response is observed in fewer than 10% of patients.
Treatment stratification for protocols of the
Berlin-Frankfurt-Munster (BFM) clinical trials group is partially
based on early response to the 7-day prednisone prophase
(administered immediately prior to the initiation of multiagent
remission induction).
[0263] Patients with no circulating blasts on day 7 have a better
outcome than those patients whose circulating blast level is
between 1 and 999/.mu.L.
Peripheral Blood Response to Multiagent Induction Therapy.
[0264] Patients with persistent circulating leukemia cells at 7 to
10 days after the initiation of multiagent chemotherapy are at
increased risk of relapse compared with patients who have clearance
of peripheral blasts within 1 week of therapy initiation. Rate of
clearance of peripheral blasts has been found to be of prognostic
significance in both T-cell and B-lineage ALL.
Induction Failure.
[0265] The vast majority of children with ALL achieve complete
morphologic remission by the end of the first month of treatment.
The presence of greater than 5% lymphoblasts at the end of the
induction phase is observed in up to 5% of children with ALL.
Patients at highest risk of induction failure have one or more of
the following features:
[0266] T-cell phenotype (especially without a mediastinal
mass).
[0267] B-precursor ALL with very high presenting leukocyte
counts.
[0268] 11q23 rearrangement.
[0269] Older age.
[0270] Philadelphia chromosome.
[0271] In a large retrospective study, the OS of patients with
induction failure was only 32%. However, there was significant
clinical and biological heterogeneity. A relatively favorable
outcome was observed in patients with B-precursor ALL between the
ages of 1 and 5 years without adverse cytogenetics (MLL
translocation or BCR-ABL). This group had a 10-year survival
exceeding 50%, and SCT in first remission was not associated with a
survival advantage compared with chemotherapy alone for this
subset. Patients with the poorest outcomes (<20% 10-year
survival) included those who were aged 14 to 18 years, or who had
the Philadelphia chromosome or MLL rearrangement. B-cell ALL
patients younger than 6 years and T-cell ALL patients (regardless
of age) appeared to have better outcomes if treated with allogeneic
SCT after achieving complete remission than those who received
further treatment with chemotherapy alone."
[0272] The term "patient" or "subject" is used throughout the
specification within context to describe an animal, generally a
mammal and preferably a human, to whom treatment, including
prophylactic treatment, according to the present invention is
provided. For treatment of symptoms which are specific for a
specific animal such as a human patient, the term patient refers to
that specific animal.
[0273] The term "cancer" is used throughout the specification to
refer to the pathological process that results in the formation and
growth of a cancerous or malignant neoplasm, i.e., abnormal tissue
that grows by cellular proliferation, often more rapidly than
normal and continues to grow after the stimuli that initiated the
new growth cease. Malignant neoplasms show partial or complete lack
of structural organization and functional coordination with the
normal tissue and most invade surrounding tissues, metastasize to
several sites, and are likely to recur after attempted removal and
to cause the death of the patient unless adequately treated.
[0274] As used herein, the term "neoplasia" is used to describe all
cancerous disease states and embraces or encompasses the
pathological process associated with malignant hematogenous,
ascitic and solid tumors. Representative cancers include, for
example, stomach, colon, rectal, liver, pancreatic, lung
(especially non-small cell lunger cancer), breast, cervix uteri,
corpus uteri, ovary, prostate, testis, bladder, renal, brain/CNS,
head and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma,
multiple myeloma, leukemia, melanoma, non-melanoma skin cancer,
acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's
sarcoma, small cell lung cancer, bone cancer, choriocarcinoma,
rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia,
mouth/pharynx, oesophagus, larynx, kidney cancer and lymphoma,
among others, which may be treated by one or more compounds
according to the present invention. Cancer which may be treated
preferentially using compositions and/or methods which employ
targeting peptides as otherwise disclosed herein include those
cancers which express CRLF-2 receptors in an upregulated manner,
including overexpression or hyperexpression of CRLF-2. Although the
principal focus of the present application is on acute
lymophoblastic leukemia (ALL) and in particular, B-cell ALL
(B-ALL), any cancer which expresses CRLF-2 in an upregulated manner
(including overexpression/hyperexpression) may be treated pursuant
to the present invention.
[0275] The term "tumor" is used to describe a malignant or benign
growth or tumefacent.
[0276] The term "additional anti-cancer compound", "additional
anti-cancer drug" or "additional anti-cancer agent" is used to
describe any compound (including its derivatives) which may be used
to treat cancer. The "additional anti-cancer compound", "additional
anti-cancer drug" or "additional anti-cancer agent" can be a
tyrosine kinase inhibitor that is different from a tyrosine kinase
inhibitor which has been previously administered to a subject. In
many instances, the co-administration of another anti-cancer
compound results in a synergistic anti-cancer effect.
[0277] Exemplary anti-cancer compounds for co-administration
according to the present invention include anti-metabolites agents
which are broadly characterized as antimetabolites, inhibitors of
topoisomerase I and II, alkylating agents and microtubule
inhibitors (e.g., taxol), as well as, EGF kinase inhibitors (e.g.,
tarceva or erlotinib) or ABL kinase inhibitors (e.g. imatinib).
Anti-cancer compounds for co-administration also include, for
example, Aldesleukin; Alemtuzumab; alitretinoin; allopurinol;
altretamine; amifostine; anastrozole; arsenic trioxide;
Asparaginase; BCG Live; bexarotene capsules; bexarotene gel;
bleomycin; busulfan intravenous; busulfan oral; calusterone;
capecitabine; carboplatin; carmustine; carmustine with Polifeprosan
20 Implant; celecoxib; chlorambucil; cisplatin; cladribine;
cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine;
dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicin
liposomal; daunorubicin, daunomycin; Denileukin diftitox,
dexrazoxane; docetaxel; doxorubicin; doxorubicin liposomal;
Dromostanolone propionate; Elliott's B Solution; epirubicin;
Epoetin alfa estramustine; etoposide phosphate; etoposide (VP-16);
exemestane; Filgrastim; floxuridine (intraarterial); fludarabine;
fluorouracil (5-FU); fulvestrant; gemtuzumab ozogamicin; gleevec
(imatinib); goserelin acetate; hydroxyurea; Ibritumomab Tiuxetan;
idarubicin; ifosfamide; imatinib mesylate; Interferon alfa-2a;
Interferon alfa-2b; irinotecan; letrozole; leucovorin; levamisole;
lomustine (CCNU); meclorethamine (nitrogen mustard); megestrol
acetate; melphalan (L-PAM); mercaptopurine (6-MP); mesna;
methotrexate; methoxsalen; mitomycin C; mitotane; mitoxantrone;
nandrolone phenpropionate; Nofetumomab; LOddC; Oprelvekin;
oxaliplatin; paclitaxel; pamidronate; pegademase; Pegaspargase;
Pegfilgrastim; pentostatin; pipobroman; plicamycin; mithramycin;
porfimer sodium; procarbazine; quinacrine; Rasburicase; Rituximab;
Sargramostim; streptozocin; surafenib; talbuvidine (LDT); talc;
tamoxifen; tarceva (erlotinib); temozolomide; teniposide (VM-26);
testolactone; thioguanine (6-TG); thiotepa; topotecan; toremifene;
Tositumomab; Trastuzumab; tretinoin (ATRA); Uracil Mustard;
valrubicin; valtorcitabine (monoval LDC); vinblastine; vinorelbine;
zoledronate; and mixtures thereof, among others.
[0278] The term "coadminister" "co-administration" or "combination
therapy" is used to describe a therapy in which at least two active
compounds in effective amounts are used to treat cancer or another
disease state or condition as otherwise described herein at the
same time. Although the term co-administration preferably includes
the administration of two active compounds to the patient at the
same time, it is not necessary that the compounds be administered
to the patient at the same time, although effective amounts of the
individual compounds will be present in the patient at the same
time.
[0279] Co-administration of two or more anticancer agents will
often result in a synergistic enhancement of the anticancer
activity of the other anticancer agent, an unexpected result. One
or more of the present formulations may also be co-administered
with another bioactive agent (e.g., antiviral agent,
antihyperproliferative disease agent, agents which treat chronic
inflammatory disease, among others as otherwise described
herein).
[0280] In one embodiment, the present invention is directed to high
surface area (i.e., greater than about 600 m.sup.2/g, preferably
about 600 to about 1,000-1250 mg.sup.2/g), preferably monodisperse
spherical silica or other biocompatible material nanoparticles
having diameters falling within the range of about 0.05 to 50
.mu.m, preferably about 1,000 nm or less, more preferably about 100
nm or less, 10-20 nm in diameter, a multimodal pore morphology
comprising large (about 1-100 nm, preferably about 2-50 nm, more
preferably about 10-35 nm, about 20-30 nm) surface-accessible pores
interconnected by smaller internal pores (about 2-20 nm, preferably
about 5-15 nm, more preferably about 6-12 nm) volume, each
nanoparticle comprising a lipid bilayer (preferably a phospholipid
bilayer) supported by said nanoparticles (the phospholipic bilayer
and silica nanoparticles together are labeled "protocells"), to
which is bound at least one antigen which binds to a CRLF-2 and/or
CD19 targeting polypeptide or protein on a cell to which the
protocells are to be targeted, wherein the protocells further
comprise (are loaded) with a small molecule anticancer agent and/or
a macromolecule selected from the group consisting of a short
hairpin RNA (shRNA), small interfering RNA (siRNA) or a polypeptide
toxin (e.g. ricin toxin A-chain or other toxic polypeptide).
[0281] Small molecule anticancer agents and macromolecules (shRNAs,
siRNAs other micro RNAs and polypeptide/proteins toxins) as
otherwise described herein may be loaded by adsorption and/or
capillary filling of the pores of the particle core. While the
nanoparticles according to the present invention are preferably
comprised of silica, they may be comprised of other materials
organic or inorganic including (in addition to the preferred
silica), alumina, titania, zirconia, polymers (e.g., polystyrene,
polycaprolactone, polylactic and/or polyglycolic acid, etc.) or
combinations thereof. In addition, the porous particles according
to the present invention may also include inorganic particles,
hydrogel particles or other suitable particles which may be added
to influence the loading of the particle and/or the release of
actives from the particle upon delivery in a biological system. In
preferred embodiments, the porous particle core includes mesoporous
silica particles which provide biocompatibility and nanoporosity.
Nanoparticles pursuant to the present invention are otherwise
described in PCT/US2010/020096, published as WO 21010/078569 on
Jul. 8, 2011, which is incorporated by reference in its entirety
herein. Mesoporous silica particles for use in the present
invention may be preferred.
[0282] The term "CRLF-2 binding peptide" is used to describe any
one or more of the peptides which are set forth in 3 or FIGS. 10-14
or equivalents thereof or as otherwise described herein. The term
CD19 binding peptide is used to describe any one or more of the
peptides which bind to CD19. The term CRLF-2 binding peptide is
directed to peptides which consist essentially of/include the
following specific peptides: MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ
ID NO:5), AAQTSTP (SEQ ID NO:6), TDAHASV (SEQ ID NO:7), FSYLPSH
(SEQ ID NO: 8), YTTQSWQ (SEQ ID NO:9), MHAPPFY (SEQ ID NO:10),
AATLFPL (SEQ ID NO:11), LTSRPTL (SEQ ID NO:12), ETKAWWL (SEQ ID
NO:13), HWGMWSY (SEQ ID NO:14), SQIFGNK (SEQ ID NO:15), SQAFVLV
(SEQ ID NO:16), WPTRPWH (SEQ ID NO:17), WVHPPKV (SEQ ID NO:18),
TMCIYCT (SEQ ID NO:19), ASRIVTS (SEQ ID NO:20), WTGSYRW (SEQ ID
NO:21) and NILSLSM (SEQ ID NO:22). Preferred CRLF-2 binding
peptides include MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID NO:5),
AAQTSTP (SEQ ID NO:6), MHAPPFY (SEQ ID NO:10), ETKAWWL (SEQ ID
NO:13), SQIFGNK (SEQ ID NO:15), AATLFPL (SEQ ID NO:11), TDAHASV
(SEQ ID NO:7) and FSYLPSH (SEQ ID NO: 8). More preferably, the
CRLF-2 binding peptide is MTAAPVH (SEQ ID NO: 4), LTTPNWV (SEQ ID
NO:5), AAQTSTP (SEQ ID NO:6) or MHAPPFY (SEQ ID NO:10). Often, the
CRLF-2 binding peptide used in embodiments according to the present
invention includes MTAAPVH (SEQ ID NO: 4) and LTTPNWV (SEQ ID
NO:5). Most often, the CRLF-2 binding peptide is MTAAPVH (SEQ ID
NO: 4). Each of these polypeptides can be conjugated or otherwise
covalently linked/complexed to protocells (for example, by
modification of the peptide through insertion of a cysteinyl
residue which can be reacted with a crosslinking agent as otherwise
described herein or a hexameric histidine oligopeptide which can be
complexed with an appropriately modified phospholipid which can
complex copper and/or nickel to which the oligopeptide will
bind).
[0283] As discussed above, each of these peptides may be
conjugated/crosslinked to a protocell as otherwise described herein
(preferably, to the phospholipid bilayer of the protocell) or
inserted as a heterologous peptide into the peptide sequence of a
bacteriophage coat polypeptide (which forms VLP's hereunder). In
certain embodiments, as otherwise described herein, a hexameric
histidine oligopeptide, a cysteinyl residue or is
complexed/covalently linked through a spacer to the binding
peptide. Generally, the spacer is between one and three amino acid
residues (such as glycine, alanine that is non-functional but can
provide spacing between the binding peptide and the group which
assists in covalently linking/complexing the binding peptide to the
protocell) in length inserted onto the carboxylic acid end of the
peptide. The spacer allows the insertion of a functional group such
as a cysteinyl residue or hexameric histidine oligopeptide which
can assist in anchoring the binding peptide to the protocell.
[0284] Additional CRLF-2 binding sequences include consensus
binding sequences which appear in an FIGS. 3 and 10-14 hereof, and
include consensus peptide sequence WPTXPW[-H] (SEQ ID NO:25),
---S[FW][ST]XWXX--WX------ (SEQ ID NO:26),
------XSPXXWXXXXX-------- (SEQ ID No:27), FS--YLP[-S][-H] (SEQ ID
NO: 34) and MT-AAP[VFW]H (SEQ ID NO:35),
[0285] It is noted that in certain instances the peptide contains
unidentified amino acids, indicated as a dash (-) or an X in the
peptide. In each instance, the unidentified amino acid may be
substituted with any amino acid without affecting binding,
preferably a small, neutral amino acid such as an alanine, glycine,
etc., among others. A consequence sequence was generated for each
group of binding peptides. A consensus sequence is a way of
representing the results of a multiple sequence alignment, where
related sequences are compared to each other, and similar
functional sequence motifs are found. The consensus sequence shows
which residues are conserved (are always the same), and which
residues are variable.
[0286] In certain embodiments, the porous particle core may be
hydrophilic and can be further treated to provide a more
hydrophilic surface in order to influence pharmacological result in
a particular treatment modality. For example, mesoporous silica
particles according to the present invention can be further treated
with, for example, ammonium hydroxide or other bases and hydrogen
peroxide to provide significant hydrophilicity. The use of amine
containing silanes such as
3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEPTMS),
among others, may be used to produce negatively charged cores which
can markedly influence the cargo loading of the particles. Other
agents may be used to produce positively charged cores to influence
in the cargo in other instances, depending upon the physicochemical
characteristics of the cargo.
[0287] In certain preferred embodiments, the lipid bilayer
comprises a phospholipid selected from the group consisting of
phosphatidyl choline, 1,2-Dioleoyl-3-Trimethylammonium-propane
(DOTAP), 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)
or mixtures thereof. In addition to a phospholipid, including the
specific phospholipids as otherwise described herein, the lipid
bilayer may also comprise cholesterol (for structural integrity of
the lipid bilayer) as well as polyethylene glycol
lubricants/solvents (e.g. PEG 2000, etc.) to provide flexibility to
the lipid bilayer. In addition to fusing a single phospholipid
bilayer, multiple bilayers with opposite charges may be fused onto
the porous particles in order to further influence cargo loading,
sealing and release of the particle contents in a biological
system.
[0288] In certain embodiments, the lipid bilayer can be prepared,
for example, by extrusion of hydrated lipid films through a filter
of varying pore size (e.g., 50, 100, 200 nm) to provide filtered
lipid bilayer films, which can be fused with the porous particle
cores, for example, by pipette mixing or other standard method.
[0289] In various embodiments, the protocell (nanoparticle to which
a lipid bilayer covers or is otherwise fused to the particle) can
be loaded with and seal macromolecules (shRNAs, siRNAs and
polypeptide toxins) as otherwise described herein, thus creating a
loaded protocell useful for cargo delivery across the cell
membrane
[0290] In preferred aspects of the present invention, the
protocells provide a targeted delivery through conjugation of
certain CRLF-2 and/or CD19 targeting peptides onto the protocell
surface, preferably by conjugation to the lipid bilayer surface.
These peptides may be synthesized with C-terminal cysteine residues
and conjugated to one or more of the phospholipids (especially,
DOPE, which contains a phosphoethanolamine group) which comprise
the lipid bilayer.
[0291] The invention is illustrated further in the following
non-limiting examples.
Example 1
Protocells and VLPs as a Potential Treatment for ALL
[0292] Here we report the use of protocells and VLPs as a potential
treatment for ALL, as well as other cancers. We have identified
peptides that effectively bind CRLF-2, a receptor that has been
found to be over-expressed in ALL, allowing for effective
preferential targeting of the protocell to leukemic cells. This has
been accomplished by affinity selection against either recombinant
CRLF-2 or BaF3 cells that were transfected to over express CRLF-2.
A variety of drugs has also been delivered to the cells, including
(high potency, high toxicity) or AG490 (low toxicity, low potency).
The in vitro results suggest that protocells are several order of
magnitude more effective at combating this leukemia than free drugs
alone. In-vivo experiments using SCID mice injected intravenously
with BaF3/CRLF2 cells are currently underway.
Protocell--Flexible Platform for Targeted Delivery
[0293] TEM image shows porous nanoparticle can serve as a support
for lipid bilayers, which in turn seal contents within the
core.
[0294] Protocells combine a high capacity for disparate cargos with
a modifiable, biocompatible surface.
Identification of Targeting Peptides
[0295] Phage display biopanning can be used to identify peptides
with high specific binding to target cells and minimal non-specific
binding.
[0296] After DNA sequencing is performed on 40 different clones
identified via biopanning to selectively bind to target leukemia
cells, Mimox software is used to align and determine consensus
sequences. Clones from these groups are then used to evaluate
binding constants.
[0297] Flow cytometry is used to evaluate binding of individual
phage clones within consensus sequences. In this case, a saturable
binding curve can be constructed, allowing for the determination of
the dissociation constant (Kd) of phage displaying potentially
specific peptides that bind cells with high levels of CRLF2
expression but not parental cell lines with minimal expression.
[0298] Following biopanning, sets of peptides can be grouped using
alignment and determination of consensus sequences.
Targeted Internalization of Protocells by Leukemia
[0299] 1. Protocells bind to target cells with high specificity at
low peptide densities due to fluid supported bilayer. 2. Become
internalized via receptor mediated endocytosis utilizing
internalization sequences. 3. Endosomal conditions facilitate
dissociation of the supported lipid bilayer while endosomolytic
peptide promotes endosomal escape and subsequent cargo release into
cytosol. 4. Cargo can be delivered to the nucleus by modification
with a Nuclear Localization Signal (NLS).
[0300] Once a targeting peptide has been selected, human cells
known to overexpress CRLF-2 (MHH CALL 4) are used to evaluate the
binding constants of peptide that has been cross-linked to
protocell-supported lipid bilayer (SLB). We find that we can obtain
high specific binding (low Kd values) with low numbers of peptides
when peptides are displayed on fluid lipids (DOIDA) within fluid
bilayers (DOPC) as a result of the ability for peptides to be
recruited to the binding site. When the targeting peptide is
displayed on a non-fluid lipid (DSIDA) within a non-fluid bilayer
(DSPC), an increased concentration of targeting peptide is required
to obtain similar specific binding. Both of these combinations
result in peptides being evenly distributed over the surface of the
protocell SLB. DOIDA in DOPC DSIDA in DSPC SLB.
[0301] The same targeting peptide can also be displayed on
protocell SLBs featuring mixtures of lipids which will form
segregated domains which serve to increase the local concentration
of peptide. When peptide is displayed on fluid lipids (DOIDA)
within a non-fluid bilayer (DSPC), we see increased binding
affinity (lower Kd) and lower peptide concentrations. This is due
to the ability for peptides displayed on fluid lipid domains to be
specifically be recruited to binding sites following concentration
within the non-fluid bulk domain. Slightly decreased binding
affinities are observed when peptides are displayed on non-fluid
lipid domains (DSIDA) within fluid SLBs (DOPC), although overall
affinities are quite high due to the increased local concentration
of peptides resulting from domain formation. This further
demonstrates the importance of local fluidity for optimal
peptide-target interactions.
[0302] Targeted protocells can become internalized within target
cells (MHH CALL 4, Mutz-5 and BaF3/CRLF-2) over time. However,
overall internalization efficiency is only .about.20%, which could
lead to non-specific cytotoxicity.
[0303] Targeted protocells featuring peptides displayed on a fluid
lipid (DOIDA) domain within a non-fluid SLB (DSPC) show high
binding affinity to target cells which over-express CRLF-2 (MHH
Call 4, Mutz-5, and BaF3/CRLF-2). Minimal non-specific binding to
control cells is seen. Non-specific binding of non-targeted
protocells is also minimal.
[0304] Targeted protocells can become internalized within target
cells (MHH CALL 4, Mutz-5 and BaF3/CRLF-2) over time. However,
overall internalization efficiency is only .about.20%, which could
lead to non-specific cytotoxicity.
[0305] Targeted protocells that are modified with R8 peptides,
known to facilitate macropinocytosis, become internalized within
target cells (MHH CALL 4, Mutz-5 and BaF3/CRLF-2) over time at over
90% efficiency.
[0306] Additionally, targeted protocells also displaying the R8
peptides show increased internalization kinetics, exhibiting
internalization half-lives to targeted cells over 5.times. shorter
than targeted protocells without R8 peptide. When coupled with the
increased internalization efficiency conferred by the addition of
the R8 peptide, non-specific cytotoxity should be minimized.
Targeted Protocells Selectively Kill Cancer
[0307] CRLF2-specific protocells loaded with the chemotherapeutic
agent, doxorubicin (DOX) induce apoptosis of CRLF2-positive cells
(MHH CALL 4) but not CRLF-2 negative cells (MOLT 4); apoptotic
cells are labeled with Alexa Fluor 647-labeled annexin V and the
cell impermeant nuclear stain, Sytox Green in the conflocal
fluorescence microscopy images. Protocells modified with the
CRLF2-specific targeting peptide and the R8 peptide result in a
low-level of non-specific cytotoxicity to CRLF2-negative cells
(NALM-6, MOLT 4, Jurkat, and parental BaF3), however, as evidenced
by LC90 values.
Minimizing Non-Specific Cytotoxicity
[0308] The CRLF2-specific peptide, MTAAPVH, does not promote rapid
internalization of protocells by CRLF2-positive cells. Therefore,
we further modified protocells with the R8 peptide, which promotes
non-specific macropinocytosis in a density-dependent fashion. The
following strategies mitigate the undesired toxicity of DOX-loaded
protocells modified with both the CRLF2-specific peptide and the R8
peptide to CRLF2-negative cells: (1) alter R8 density to decrease
the amount of time required for CRLF2-positive cells to internalize
surface-bound protocells and (2) attempt to select for
CRLF2-specific peptides that promote internalization by
CRLF2-positive cells upon protocell binding.
Experimental Details
[0309] FIG. 1 illustrates how a protocell is a flexible platform
for targeted delivery. The TEM image shows that a porous
nanoparticle can serve as a support for lipid bilayers, which in
turn seal contents within the core. The TEM image shows that a
porous nanoparticle can serve as a support for lipid bilayers,
which in turn seal contents within the core.
[0310] The original peptides used in these experiments were
identified using the process of phage display. In this process,
complex library of phage displaying random peptide sequences is
allowed to bind to a cell-based selection target. Phage display
biopanning was used to identify peptides with high specific binding
to target cells and minimal non-specific binding. Unbound phage are
washed away and bound phage are eluted and used to infect bacteria
for amplification. This process can be carried out iteratively
until a population of phages that tightly bind the target is
obtained. Affinity selection via phage display is shown in FIG. 2.
The left side of FIG. 2 is a schematic depicting affinity selection
using a filamentous phage library (7-mer peptide sequences).
Conditions used in affinity selection are described in the right
side of FIG. 2.
[0311] A library of filamentous phage displaying a complex random
library of peptides is created and allowed to bind to cells
displaying the target surface marker. The sample is washed and the
bound phage are eluted and subjected to negative selection against
a parental cell line lacking the target surface marker. Bacterial
cells are then infected with the reduced library for amplification
and the process is repeated iteratively until an enriched
population of binding phage is acquired.
[0312] The selections were carried out on BaF3 cells that had been
induced to create the cell receptor CRLF2 and express it on their
surface. Negative selections were carried out on the parental cell
line (no CRLF2) in order to assure that the ligand identified was
in fact binding to the desired target and not to other features on
the surface of the cells.
[0313] Three peptide sequences were pulled from the enriched
population of peptides that exhibited binding to CRLF2: TDAHASV,
FSYLPSH, and MTAAPVH (best binder).
Virus-Like Particles and Virus-Like Particles as a Display
Platform
[0314] A recently developed alternative to phage display involves
the use of Virus-Like Particles or VLPS. VLPs of the bacteriophage
MS2 are constructed of 90 fused dimers That self-assemble into an
icosahedral shell 23 nm in diameter. These perfectly monodisperse
particles can be engineered to display a complex random library of
peptides on their surfaces in either constrained (inserted into the
A-B loop) or non-constrained (inserted at the N-terminus)
conformation. Unlike the phage these particles are modeled after,
VLPs are non-infectious. When they self-assemble, they encapsulate
their own RNA. This not only allows for a mechanism of introducing
cargo, but allows for particles with specific binding affinity to
be isolated, the RNA extracted, reverse transcribed and amplified
to allow for the production of more particles displaying that
specific peptide (see FIG. 3). VLPs can be made using the
techniques described in Hooker J M, Kovacs E W, Francis M B.
Interior surface modification of bacteriophage MS2. J Am Chem Soc.
2004; 126:3718-9, or they can be made using techniques that are
either well-known to those of ordinary skill in the art or as
otherwise described herein.
[0315] More specifically, the MS2 viral shell is built from 90 coat
protein dimers that, when expressed from a plasmid in E. coli,
spontaneously self-assemble into an icosahedral shell of, e.g. 27.5
nm diameter. Two modes of display are possible. Foreign peptides
are displayed by inserting them into coat protein's AB-loop.
However, folding of the wild type coat protein does not generally
tolerate such insertions and we found it necessary to engineer a
more stable molecule. Taking advantage of the physical proximity in
the dimer of the N-terminus of one subunit to the C terminus of the
other, we duplicated the coat protein coding sequence and fused the
two copies into a single reading frame, so that both halves of the
dimer are produced as a single polypeptide. Covalently tethering
the two halves monomers to one another greatly increased the
protein's stability and dramatically improved its tolerance of
foreign peptides inserted into the AB-loop of the downstream half
of this "single-chain dimer".
[0316] We find that in excess of 90% of clones with 6-mer, 8-mer,
or 10-mer random peptide insertions yield properly assembled VLPs,
each displaying 90 copies of a foreign peptide on its surface.
Protein display is accomplished by genetically fusing a foreign
sequence to one of coat protein's termini (usually the C-terminus).
However, the presence of a fusion on every copy of coat protein may
interfere with capsid assembly. Therefore, our strategy is to fuse
the foreign protein to the C-terminus of coat protein with a stop
codon between. A nonsense-suppressing tRNA causes occasional read
through of the stop codon, and production of the fusion protein.
Suppression is relatively inefficient, so that only a few percent
of coat protein molecules actually contain the C terminal
extension. Co-assembly of the wild-type and fusion proteins
produces a VLP with an average of a few foreign molecules per
particle. It is also possible to fuse the foreign protein directly
to coat, without an intervening stop codon. In that case the fusion
and non-fusion versions of the protein are separately produced and
co-assembled into VLPs in vitro. In this proposal we describe
display of single-chain antibodies (scFv's) for the B-cell specific
surface antigens, CD19 and CD22, and of the CRLF2-specific ligand,
thymic stromal lymphopoietin (TSLP) by this method.
[0317] It is important to note that each VLP encapsidates its own
mRNA. This means that the nucleotide sequences encoding any
particular VLP and its guest peptide or protein are contained
within the particle itself, and can be recovered by reverse
transcription and polymerase chain reaction, making possible the
affinity selection scheme illustrated in FIG. 2. Random sequence
peptide libraries, for example, can be subjected to bio-panning on
any arbitrarily chosen target. Amplification and re-cloning of the
selected sequences leads to the identification of peptide ligands
specific for the target. To facilitate library construction and
screening, we constructed a plasmid vector (pDSP62) that expresses
coat protein at high levels from the bacteriophage T7 promoter. It
confers resistance to kanamycin and normally replicates using a
ColE1 origin. It also contains a M13 replication origin so that a
single-stranded version of the plasmid can be produced after
super-infection with an M13 helper phage. This allows the
straight-forward production of complex random sequence peptide
libraries by extension in vitro of mutagenic primers on circular
single stranded templates using the efficient mutagenesis procedure
of Kunkel et al., Rapid and efficient site-specific mutagenesis
without phenotypic selection. Proc Natl Acad Sci USA 1985,
82:488-492.
[0318] To restrict the insertion of peptides to the AB-loop of the
downstream half of the single-chain dimer, the upstream copy is a
synthetic "codon-juggled" coat sequence containing the maximum
possible number of silent mutations. Thus, mutagenic primers can be
targeted to anneal specifically to the downstream site. Using this
vector, random sequence timer, 7mer, 8mer and 10 mer libraries
containing more than 1010 individual members have been produced.
The high density of MS2 VLP display (90 peptides per particle) can
make it difficult during affinity selection to discriminate
peptides with high intrinsic binding affinities from those that
have low affinity, but bind with high avidity by virtue of multiple
weak interactions. To introduce valency control in the MS2 system
we made an alternate version of the sc-dimer with a stop codon
separating its two halves (in pDSP62(am)). This mutant normally
produces only wild-type coat protein from its upstream half, but in
the presence of a nonsense suppressor tRNA, a small percentage of
ribosomes read through the stop codon to produce the entire
sc-dimer with its guest peptide (remembering that the peptide is
present only in the downstream half). Both the wild-type and
sc-dimer proteins are synthesized from a single mRNA, which they
encapsidate when they co-assemble into a mosaic VLP that displays
about three peptides per particle on average. Using the MS2 VLP
system three or four rounds of affinity selection against
antibodies with known epitopes (e.g. the anti-Flag antibody, M2)
yield peptides that closely mimic those epitopes.
Cell-Based Targets for Affinity Selection of CRLF2-Binding
Peptides.
[0319] Affinity selections on cellular targets are complicated by
the heterogeneity of surface protein expression on mammalian cells,
which frequently results in a large number of peptides that bind
unsuitable targets. A major goal of the work we propose is to
create an efficient selection/counter-selection scheme. To
illustrate our strategy, the gene encoding human CRLF2 was
introduced into BaF3 cells (a murine IL3-dependent pro-B cell line)
and the protein's abundant surface expression was confirmed by FACS
(as shown in Project 2). The resulting BaF3-CRLF2 cells, together
with the BaF3 parental line (which specifically lacks CRLF2),
served as a matched selection/counter-selection pair and provided a
convenient means of discarding affinity selectants that bind the
many non-CRLF2 receptors inevitably encountered on the BaF3
surface. Affinity selections conducted in our laboratories using
commercial M13 display libraries have identified a peptide ligand
to CRLF2 (TDAHASV), which is shown in FIG. 3 to mediate the binding
of M13 phage selectants to BaF3-CRLF2 with an apparent Kd of about
3 nM, while not showing significant binding to the BaF3 parent. As
shown in Project 2, this CRLF2 targeting peptide has already been
conjugated to protocells and we have demonstrated selective binding
and toxicity in CRLF2-expressing ALL cell lines.
Targeting VLPs to Specific Cell Types with scFv's.
[0320] Monoclonal antibodies specific for a wide range of cell
surface receptors represent a rich source of potential targeting
molecules. We described above a system that enables the fusion a
scFv to the C-terminus of coat protein, and which through nonsense
suppression of a stop codon separating the two sequences, permits
the simultaneous production from a single mRNA of coat protein and
the scFv fusion. Co-assembly of the two proteins produces a VLP
with an average of a few scFv molecules per particle. We have so
far fused several different scFv's to coat protein and demonstrated
the ability of the VLP-scFv to bind its target. Based on published
amino acid sequences Cheng W W, Das D, Suresh M, Allen T M:
Expression and purification of two anti-CD 19 single chain Fv
fragments for targeting of liposomes to CD19-expressing cells.
Biochim Biophys Acta 2007, 1768:21-29; Stemmer W P, Crameri A, Ha K
D, Brennan T M, Heyneker H L: Single-step assembly of a gene and
entire plasmid from large numbers of oligodeoxyribonucleotides.
Gene 1995, 164:49-53, we synthesized (using assembly PCR Peabody,
D. S. (2003) A viral platform for chemical modification and
multivalent display. J. Nanobiotech. 1: 5.) an E. coli
codon-optimized DNA sequence that encodes the anti-CD19 protein and
fused it to the C-terminus of the MS2 coat protein sequence with an
amber codon at the fusion junction. When this gene is expressed in
bacteria with a suppressor tRNA, it produces large amounts of
single-chain coat protein, and small amounts (a few percent) of the
coat-scFv fusion, which co-assemble to yield a VLP displaying a few
antibodies per particle, on average. Precise quantitation of the
relative amounts of the two proteins has not yet been carried out,
but this is one of the variables we seek to optimize with respect
to particle yield, and cell binding and internalization. FACS
analysis shows that the CD19-specific scFv directs VLPs to bind
CD19+ cells (FIG. 16(b)). Future studies will characterize the
affinity of the interaction and more carefully document its
specificity.
[0321] FIG. 3 shows how flow cytometry is used to evaluate binding
of individual phage clones within consensus sequences. In this
case, a saturable binding curve can be constructed, allowing for
the determination of the disassociation constant (K.sub.d) of phage
displaying potential specific peptides that bind cells with high
levels of CRLF2 expression but not parental cell lines with minimal
expression. Data shown for the MTAAPVH phage clone on BaF3/CRLF2
(A) and parental BaF3 cells. (B) Binding curves were constructed by
titrating the amount of phage with constant cell concentrations in
order to quantitatively describe binding. Non-specific binding was
determined by incubating wild-type phage with the same cell lines.
Specific binding to CRLF2 positive cells is significant; Specific
binding to the parental cells is extremely minimal.
[0322] As shown in FIG. 3, following biopanning, sets of peptides
can be grouped using alignment and determination of consensus
sequences. After DNA sequencing is performed on 40 different clones
identified via biopanning to selectively bind to target leukemia
cells, Mimox software is used to align and determine consensus
sequences (LEFT SIDE). Clones from these groups are then used to
evaluate specific binding constants.
[0323] Flow cytometry is used to evaluate binding of individual
phage clones within consensus sequences. In this case, a saturable
binding curve can be constructed, allowing for determination of the
dissociation constant (Kd) of phage displaying potentially specific
peptides that bind cells with high levels of CRLF2 expression but
not parental cell lines with minimal expression. (RIGHT SIDE)
[0324] VLPs are thought to be comparable to phage in their ability
to conduct selections, and have successfully identified peptides
that mapped directly onto the variable regions of specific
antibodies. In addition, they are a suitable vehicle for delivery
of cargo. Small molecule therapeutics and labels can be tagged with
the RNA pac site that triggers self-assembly and thereby
encapsulated within VLPs with relative ease.
[0325] CRLF2-targeting protocells were prepared using the
techniques described herein and included the targeting and
endosomolytic peptides and anticancer drugs ("cargo") shown in FIG.
2. As depicted in FIGS. 1 and 2, porous nanoparticles can serve as
a support for lipid bilayers, which in turn encapsulate CRLF2
and/or CD 19-targeting active ingredient(s) within the core.
[0326] The progression of this experiment aimed to identify binding
peptides via phage display and to quantify their interactions with
CRLF2 expressing cells while displayed on filamentous phage,
genetically display these original peptides on the surface of VLPs,
quantify the VLP interactions with CRLF2 expressing cells, and then
identify peptides via the VLP based affinity selection process
illustrated herein.
[0327] As shown in FIG. 4, it was determined that the protocells
bind to target cells with high specificity at low peptide densities
due to a fluid supported bilayer. FIG. 5 illustrates how once a
targeting peptide has been selected, human cells known to
over-express CRLF-2 (MMH CALL 4) were used to evaluate the binding
constants of peptide that has been cross-linked to
protocell-supported lipid bilayer (SLB). The same targeting peptide
can also be displayed on protocell SLBs featuring mixtures of
lipids which will form segregated domains which serve to increase
the local concentration of peptide. When peptide is displayed on
fluid peptides (DOIDA) within a non-fluid bilayer (DSPC), we see
increased binding affinity (lower Kd) and lower peptide
concentrations. This is due to the ability for peptides displayed
on fluid lipid domains to be specifically be recruited to binding
sites following concentration within the non-fluid bulk domain.
Slightly decreased binding affinities are observed when peptides
are displayed on non-fluid lipid domains (DSIDA) within fluid SLBs
(DOPC), although overall affinities are quite high due to the
increased local concentration of peptides resulting from domain
formation. This further demonstrates the importance of local
fluidity for optimal peptide-target interactions.
[0328] As illustrated in FIG. 6, targeted protocells can became
internalized within target cells (MMH CALL 4, Mutz-5 and
BaF3/CRLF-2) over time. The upper-right part of FIG. 6 depicts
disassociation constants for CRLF-2 targeted protocells for various
CRLF-2 positive and CRLF-2-negative cell lines. The lower portion
of FIG. 6 shows images of CRLF-2-positive (MHH CALL 4) and
CRLF-2-negative (MOLT 4) cells that were exposed to targeted
protocells at 37.degree. C. for two hours. Cells were pre-treated
with cytochalasin D to inhibit internalization of surface-bound
protocells. Bilayer composition=DOIDA in DSPC; all K.sub.d and
k(on) measurements were conducted at 37.degree. C. using cells that
had been exposed to cytochalasin D, which inhibits actin
polymerization and, therefore, inhibits clathrin- and
caveolae-dependent endocytosis, as well as macropinocytosis. Cells
are labeled with CellTracker Green and DAPI in the microscopy
images.
[0329] As shown in FIG. 6, targeted protocells featuring peptides
displayed on a fluid lipid (DOIDA) domain within a non-fluid SLB
(DSPC) show high binding affinity to target cells which
over-express CRLF-2 (MHH Call 4, Mutz-5, and BaF3/CRLF-2). Minimal
non-specific binding to control cells is seen. Non-specific binding
of non-targeted protocells is also minimal.
[0330] The on-rates of different concentrations of targeting
peptides displayed on fluid and non-fluid lipids within fluid and
non-fluid lipid bilayers are also dependent on fluidity and local
concentration. When peptides are displayed on lipids which form
domains within the SLB, targeted protocells will more quickly reach
a half-maximal level of saturated binding to target cells. This is
a consequence of increased local concentration of targeting
peptides on domains. Furthermore, if this domain remains fluid,
half-maximal saturation is reached at an even faster rate.
[0331] FIG. 6 also shows that targeted peptides displayed on a
fluid lipid (DOIDA) domain within a non-fluid SLB (DSPC) showed
high binding affinity to target cells which over-express CRF-2 (MHH
Cell 4, Mutz and BaF3/CRLF-2).
[0332] FIG. 7 also shows that targeted protocells became
internalized within target cells (MHH CALL4, Mutz-5 and
BaF3/CRLF-2) over time. More specifically, FIG. 7 illustrates that
targeted protocells that are modified with R8 peptides, known to
facilitate internalization, become internalized within target cells
(MHH CALL 4, Mutz-5 and BaF3/CRLF-2) over time at over 90%
efficiency.
[0333] The upper-left portion of FIG. 7 depicts the internalization
efficacy of CRLF-2-targeted protocells in the absence of the R8
peptide. The upper right portion of FIG. 7 presents images of
CRLF-2-positive (MHH CALL 4) and CRLF-2-negative (MOLT 4) cells
exposed to targeted protocells for 37.degree. C. for two hours.
N.D.=not detectable; bilayer composition=DOIDA in DSPC with 5 wt %
DSPE; approximate peptide density=10 targeting peptides/protocell
and .about.500 H5WYG peptides/protocell. Targeted protocells can
become internalized within target cells (MHH CALL 4, Mutz-5 and
BaF3/CRLF-2) over time. However, overall internalization efficiency
is only .about.20%, which could lead to non-specific cytotoxicity.
The lower-left portion of FIG. 7 depicts the internalization
efficacy of CRLF-2-targeted protocells in the presence of the R8
peptide. The lower right portion of FIG. 7 presents images of
CRLF-2-positive (MHH CALL 4) and CRLF-2-negative (MOLT 4) cells
exposed to targeted protocells for 37.degree. C. for two hours.
N.D.=not detectable; bilayer composition=DOIDA in DSPC with 5 wt %
DSPE; approximate peptide density=10 targeting peptides/protocell
and -500 H5WYG peptides/protocell.
[0334] FIG. 8 illustrates that targeted protocells that display the
R8 peptide showed increased internalization kinetics. The
upper-left portion of FIG. 8 depicts internalization kinetics for
CRLF-2-targeted protocells in the absence and presence of the R8
peptide. The upper-right portion of FIG. 8 depicts images of
CRLF-2-positive (MHH CALL 4) cells exposed to targeted protocells
at 37.degree. C. for twenty-four hours. The bottom of FIG. 8
presents images of CRLF-2 positive (MHH CALL 4) and CRLF-2 negative
(MOLT) that were continually exposed to 75 nM of DOX (encapsulated
within CRLF-2-targeted, R8-modified protocells for 48 hours at
37.degree. C. Bilayer composition=DOIDA in DSPC with 5 wt % DSPE;
approximate peptide density=10 targeting peptides/protocell,
.about.500 H5WYG peptides/protocell, and .about.500 R8
peptides/protocell.
[0335] Additionally, targeted protocells also displaying the R8
peptides show increased internalization kinetics, exhibiting
internalization half-lives to targeted cells over 5.times. shorter
than targeted protocells without R8 peptide. When coupled with the
increased internalization efficiency conferred by the addition of
the R8 peptide, non-specific cytotoxity should be minimized.
[0336] FIG. 9 illustrates that CRLF-2 specific protocells loaded
with the chemotherapeutic agent doxorubicin (DOX) induced apoptosis
of CRLF-2-positive cells (MHH CALL4) but not CRLF-2 negative cells
(MOLT4).
[0337] FIGS. 10 and 11 depict data for selections against
BaF3/CRLF-2 (4.degree. C.).
FIGS. 12 and 13 depict data for selections against BaF3/CRLF-2
(37.degree. C.). FIG. 14 depicts data for selections against
BaF3/CRLF-2 (37.degree. C. with trypsin), as determined in the
experiment(s) of Example(s).
Quantification of Identified Peptides on MS2 VLPs
[0338] In order to quantify the original peptides on MS2 VLPs, the
peptides first had to be genetically inserted in the coat protein
dimer. This is done by designing primers that anneal to the DNA in
a desired location and contain an insert coding for the peptide
sequence to be displayed. Through PCR, restriction digests, and
ligations, a new DNA strand, or plasmid, is produced. This plasmid
can then be transformed into E. coli and induced to produce coat
protein at a large scale. These proteins self-assemble into VLPs
that can then be isolated and used to conduct experiments.
[0339] For flow cytometry experiments, particles were labeled with
Alexa-fluor-647 and incubated with various cell types for an hour
before the samples were washed and immediately measured using a
FACSCaliber flow cytometer (data shown in FIGS. 15(1)-(8)). Samples
included both targeted protocells (displaying the targeting
peptides identified via phage display) and nontargeted protocells
(displaying a non-relevant peptide and particles not displaying any
additional peptides). These particles were screened against both
BaF3/CRLF-2 and parental BaF3 cells. As expected, none of the
samples demonstrated significant binding other than the targeted
protocells incubated with target-expressing cells (lower right
panel). To confirm binding, confocal microscopy images were taken
of these samples as well.
[0340] Virus-like particle based affinity selection can be
conducted using techniques similar to the phage display described
above and depicted in FIG. 3, although VLPs are non-infectious. The
RNA must be isolated from eluted VLPs, reverse transcribed into
DNA, amplified, re-inserted into a plasmid encoding for coat
protein which was then transformed into bacteria for production of
particles.
Validation of Display Platform by Targeting EGFR
[0341] Issues arose with trying to conduct VLP-based affinity
selection on the cell expressing CRLF-2. Due to differences between
the way that CRLF2 is displayed on the surface of naturally
expressing cells and the way it is presented on the BaF3/CRLF2 cell
line, as well as an incomplete knowledge of the receptor, it was
difficult to determine if obstacles in the course of the research
were due to flaws in the protocol itself, or in the presentation of
the target. To this end, it was decided to proceed with VLP-based
affinity selection on a better understood target: Epidermal Growth
Factor Receptor (EGFR). Not only is EGFR well understood, it is
clinically relevant. Anti-EGFR antibodies are currently being used
to treat several varieties of cancer. Also, a new approach is
required because treatments with these anti-bodies are beginning to
lose effectiveness, and some studies suggest might activate the
receptor leading to increased tumor motility. Selections are
conducted against EGFR protein using a mixed library of VLPs
displaying peptides of 6, 7, 8, and 10 amino acids in length. Prior
to selection, the EGFR is affinity captured onto the surface of a
microcell plate via a GST-tag. This increases not only the amount
of protein adsorbed to the well, but also orients the proteins in
such a manner as to increase the statistical likelihood of
selecting for peptides that bind in the receptor binding pocket.
Selections are currently in the middle of the third iteration of
positive selection (selection against EGFR) and have undergone one
round of negative selection (to reduce the number of VLPs in the
propagated library that are binding to the glutathione on the
surface of the wells). Comparison run during the negative
selections confirm that the enriched library does include VLPs that
selectively bind to EGFR.
[0342] A further experiment assessed the ability of a targeting
peptide to direct the binding of a virus-like particle to
CRLF2-producing cells, one was fused to the N-terminus of
bacteriophage MS2 coat protein. The virus-like particle (VLP) thus
produced was assayed for its ability to bind cells producing the
targeted receptor. FIG. 15(9) shows the structure of a plasmid that
expresses the MS2 coat protein single-chain dimer with a fusion of
a CRLF2 targeting peptide (TDAHASV SEQ ID NO:7) at its N-terminus.
This protein was produced from the plasmid in bacteria, where it
spontaneously assembled into a VLP displaying 90 copies of the
targeting peptide on its surface. To assess the particle's ability
to bind cells with surface expressed CRLF2, two different cell
lines (REH and BaF3) were stably transformed with the CRLF2 gene,
thus producing REH-CRLF2 and BaF-CRLF2. Each of the parental cell
lines expresses no CRLF2, but the derivatives express it
abundantly. Purified VLPs were incubated with the various cell
types, which were then treated with a fluorescently labeled
antibody specific for MS2 coat protein. FACS analysis reveals the
ability of the targeted VLPs to specifically bind only the cells
producing CRLF2 (FIG. 15(10)).
Example 2
CD19 Protocol
[0343] CD19 IgG1 was partially reduced via reaction with a 60-fold
molar excess of TCEP for 20 minutes at room temperature. Reduced
antibody was then desalted and incubated with protocells (DOPC with
30 wt % cholesterol and 10 wt % maleimide-PEG-DMPE) overnight at 4
C. Protocells were washed 3.times. with PBS before being added to
cells.
[0344] For flow cytometry experiments, particles were labeled with
Alexa-fluor-647 and incubated with various cell types for an hour
before the samples were washed and immediately measured using a
FACSCaliber flow cytometer (data shown in FIG. 16). Samples
included both targeted VLPs (displaying the targeting peptides
identified via phage display) and nontargeted VLPs (displaying a
non-relevant peptide and particles not displaying any additional
peptides). These particles were screened against both BaF3/CRLF-2
and parental BaF3 cells. As expected, none of the samples
demonstrated significant binding other than the targeted VLPs
incubated with target-expressing cells (lower right panel).
Example 3
N2.v.1 ALL--a Model System to Understand and Perfect Targeted
Delivery
[0345] Diagnostic leukemic blast samples were obtained from 207 ALL
patients enrolled in Children's Oncology Group (COG) trial 9906.
These children had characteristics (older age and higher white
blood count) that suggested that they were at an elevated risk of
relapse (44% event free survival in earlier trials). RNA was
extracted. Biotinylated cRNA was synthesized and hybridized to
HG_U133A_Plus2.0 oligonucleotide microarrays, and fluorescent
intensity signals were obtained for 54,688 probes sets
corresponding to named genes and uncharacterized transcript). Final
intensities were obtained after a standard masking and
normalization procedure. "Outlier" genes, defined as transcripts
expressed several logs above or below the mean in a subset of
samples were identified by a variation of a COPA analysis and
unsupervised hierarchical clustering was performed (FIG. 17(a))
Even in the absence of information concerning patient
characteristics (including outcome) several clear clusters were
obtained. Remarkably, cluster 8 identified a group of children with
a markedly poor outcome (Kaplan Meier plot in FIG. 17(b)).
[0346] This genetic analysis is important for several reasons. A
current protocol under consideration by COG involves the up front
testing of diagnostic leukemic blasts using the gene signature
described above to identify a cohort of patients that are almost
certain to be unresponsive to current therapies. The long term goal
of this COG effort is to identify a new generation of treatment
specifically tailored to this extremely high risk group.
Approximately 50% of these children would be predicted to have an
activating mutation in JAK, and the use of well characterized JAK
inhibitors is also in the planning stages. However, all of these
patients express a subset of the genes used to identify the cohort,
and a targeting mechanism dependent only on the presence of the
gene products independent of any function would be an ideal
approach to new therapies.
[0347] We chose CD99 and CRLF from the list of potential cluster 8
targets as initial VLP/protocell targets for multiple reasons. The
expression of, either gene alone is predictive of a markedly poor
outcome as shown in FIG. 17(b) B, C. Both genes have a
well-characterized extracellular domain of 100-125 residues,
representing an ideal bait for the identification of binding
peptides. In addition, there are suggestions that targeting cells
that express either gene will both be tolerated by animals and have
applications in diseases other than pediatric ALL.
[0348] CD99 is expressed at high levels in multiple tumor types,
including Ewing's sarcoma. Antibody-based targeting of CD99 has
developed in animals and has been well tolerated, suggesting that
the depletion of nonmalignant cells that are CD99 positive is not a
major issue. Expression of CD99 is also elevated in cells
infiltrating atherosclerotic plaques, and vaccination of mice
against CD99 provided protection against plaque formation without
major side effects despite the long term loss of CD99 positive
lymphocytes and monocytes.
[0349] Although CRLF2 (also termed TSLPR) appears to play a minor
role in embryonic hematopoiesis, a genetic knockout does not
display a phenotype, suggesting that CRLF2 positive cells are not
required for critical functions after birth. Alterations in CRLF2
signaling have been postulated to play a major role in aberrant
inflammatory responses such as acute dermatitis and asthma, and a
significant effort is underway to find small molecule inhibitors of
CRLF2 function or compounds to deplete CRLF2 positive cells in
patients with severe allergic disorders.
[0350] We have cloned both CD99 and CRLF2 in retroviral based
expression systems, infected cultured cells that lack endogenous
expression, and selected stable transfectants. Cells infected with
the CRLF2 virus express very high levels of the protein that is
properly trafficked to the membrane, since it is accessible to
extracellular antibodies. Similar results have been obtained with
CD99. These cells will allow us to take a novel approach to the
identification of targeting peptides. Rather than performing
differential screens with normal and malignant cells as is often
done, we can use cells that differ only by the expression of a
single gene product that we have shown to be differentially
expressed on the surface of the cell that is to be targeted. We
have also made constructs containing the extracellular domain of
both CRLF2 and CD99 fused to GST, allowing for a bead based
selection strategy.
[0351] Taken together these observations suggest that pediatric ALL
is an optimal model system for the nano-based targeting
experiments. We have used gene expression arrays to characterize a
cohort of pediatric ALL patients with a dismal outcome despite
intensification of state of the art therapies. We have identified a
specific set of proteins with extracellular domains expressed in
the blasts of these patients, and propose that a novel approach in
which cytotoxic reagents are delivered to cells based on the
differential expression of these proteins may markedly improve
their survival. We also argue that based on prior studies,
targeting of cells expressing either CD99 or CRFL2 will have a
minimum of side effects, and may well have important implications
for other diseases.
Example 4
Development of Targeting Ligands
[0352] CRLF2.
[0353] CRLF2 may be targeted using its natural ligand TSLP or with
peptides that are specifically directed towards CRLF2; both
approaches are being developed. To identify CRLF2-specific
targeting peptides, we have used a commercial M13 filamentous phage
library and our new nanotechnology platform method (MS2 virus-like
peptide (VLP) displays) to screen for peptides against Ba-F3 cells
(a murine IL-3-dependent pro-B cell ALL cell line) engineered to
stably express human CRLF2. Peptides selected by affinity for
Ba-F3-CRLF2 cells were counter-selected against parental Ba-F3
cells to eliminate any phage binding receptors common to both cell
types. We find that a matched selection/counter-selection pair
greatly increases the specificity of the affinity selection
process. It is important to note that in the VLP screening method,
each VLP encapsidates its own mRNA. This means that the nucleotide
sequences encoding any particular VLP and its guest peptide or
protein are contained within the particle itself and can be
recovered by reverse transcription and polymerase chain reaction.
Amplification and re-cloning of the selected sequences leads to the
identification of peptide ligands specific for the target..sup.23
Through this approach, 12 CLRF2 targeting peptides were identified
which caused the filamentous phage selectants to bind cells at
nanomolar affinities; their specificity for CRLF2 was further
demonstrated by their ability to bind the purified protein in vitro
(data not shown). Affinity selections conducted in our laboratories
have identified a peptide ligand to CRLF2 (TDAHASV) (FIG. 18),
demonstrating a Kd of 27.9 nM with no significant binding to the
BaF3 parental line (Kd of <3 .mu.M)). This targeting peptide has
already been conjugated to protocells and we have demonstrated
selective binding and toxicity in CRLF2-expressing ALL cell
lines
Targeting CD19 with Single, Chain Variable Region Antibody
Fragments (scFV).
[0354] Monoclonal antibodies directed towards B cell-specific cell
surface antigens (such as CD 19, CD20, or CD22) represent an
additional source of targeting agents that can be exploited for
nanotherapeutic approaches against a broad range of B cell
malignances. Compared to peptides, antibodies offer the prospect of
high-affinity binding even when presented at low valency on
nanoparticles. We will use our established methods to develop
CD19-targeted protocells by conjugating to protocells the single
chain variable fragment (scFv) derived from FMC63 anti-CD 19 which
has already been successfully used to target CD 19+ cells in murine
xenograft models and in human immunotherapy clinical trials for
CLL, and more recently, ALL. CD19 is a type I transmembrane
glycoprotein of the immunoglobulin Ig superfamily with B
cell-restricted expression. As CD 19 is expressed in the earliest
(early pre-B cells) to the latest (plasma cells) stages of B cell
development, it is an attractive target for therapy of a broad
range of B cell malignancies. Numerous B cell-specific anti-CD 19
biologics, including immunoconjugates, have demonstrated efficacy
in xenograft models and in human clinical trials for various B cell
malignancies. Although CD 19 is efficiently internalized in B cells
and is more consistently expressed as a target in pre-B ALL, some
investigators believe that CD22 may be a better therapeutic target
due to its more rapid internalization. Should CD19-targeted
protocells be less than optimally internalized, we will consider
the development of CD22-targeted protocells using scFv such as
those derived from RFB4.
Example 5
CRLF2-Targeted Protocells
[0355] We synthesized CRLF2-targeted protocells by conjugation of
the CRLF2-targeting peptide TDAHASV to protocells. CRLF2-targeted
protocells were demonstrated to possess a 1000-fold higher affinity
for engineered BaF3-CRLF2 cells expressing high levels of CRLF2
(FIG. 19) and for the MUTZ5 or MHHCALL4 cells (FIG. 19, 20)
(established human ALL cells lines with CRLF2 genomic
rearrangements producing high levels of cell surface CRLF2 proteins
and JAK tyrosine kinase mutations), when compared to untargeted
protocells, the parental BAF3 cell line, or the CRLF2(-)
CD19-positive NALM6 B-precursor ALL cell line, which served as
controls. This affinity was also achievable at very low peptide
densities (FIG. 19A) due to the fluid protocell surface,
potentially minimizing non-specific binding and/or immune
responses. Targeted protocells loaded with DOX (which is
intrinsically fluorescent) were able to selectively bind to cells
expressing CRLF2, and after incubation at 37.degree. C., to become
internalized and deliver drug to the cytoplasm of the cells within
24 hours, while showing no non-specific interactions with control
cells (FIGS. 19B, 20). Further, modification of the protocell
surface with an octa-arginine (R8) peptide promoted this selective
internalization in a density-dependent manner (FIG. 19C), proving
that protocells support complex synergistic interactions enabling
targeting and internalization for cancers whose targeting peptides
might be poorly internalized. These preliminary studies demonstrate
that we can selectively target CRLF2-expressing ALL cells with
CRLF2-targeted nanocarriers in vitro, and, that the protocell and
its cargo are internalized and taken up by the cytoplasm.
Defining Optimal Therapeutic Cargos.
[0356] The ability of protocells to protect their therapeutic cargo
until released within the target cell and to deliver multiple
cargoes is being exploited initially in vitro to determine the most
efficacious drug combinations for packaging into ALL-targeted
protocells. As shown in FIG. 20, when CRLF2-targeted protocells
with encapsidated DOX were incubated with the established MHHCALL4
ALL cell line (with CRLF2 genomic rearrangements and high CRLF2
expression on the cell surface), binding, protocell and drug
uptake, and DOX release into the cytoplasm could be demonstrated in
CRLF2-expressing cells but not in controls. Although high-risk ALL
patients tend to be resistant to intensive therapeutic regimens, we
have shown in preliminary studies that after uptake and drug
delivery, CRLF2-targeted protocells with encapsidated DOX promoted
rapid apoptosis and cell death in MHH CALL4 cells (FIG. 21). Using
the established and engineered ALL cell lines described above and
in Aim 2, we are testing traditional ALL therapeutic drug
combinations as well as novel compounds that we have demonstrated
are effective against high-risk ALL (such as the signal
transduction inhibitor rapamycin which we have shown is synergistic
with DOX (see FIG. 19 in Core D and associated discussion). The
therapeutic efficacy of drugs encapsidated in targeted protocells
is being compared to exposure of the cell lines to targeted
protocells lacking therapeutic cargos, non-targeted protocells
(with and without cargos) and free drug(s) using cell biologic,
flow cytometric, and phosphoflow cytometric assays (in Core C),
allowing us to test and model pharmacodynamic assessments of target
inhibition in ALL cells in vitro. The amount of drug carried per
protocell is tunable (ranging from 0-50% by weight) and can be
modified by changing the concentration of drug in the loading
solution. The optimal concentrations of therapeutic cargos will be
defined for each drug and drug combination through iterative
interactions between in vitro and in vivo.
[0357] In our initial studies with DOX, we wish to deliver an
equivalent dose via targeted protocells as we will for free drug,
in an appropriate therapeutic dose range, in vitro and in the ALL
xenograft models in vivo. Thus, the initial loading dose for DOX in
protocells will be at 10% protocell particle weight in order to be
equivalent to the planned injected dose of free drug (0.2 mg per
mouse at 2 mg particles). These studies will include comprehensive
dose-response curves using the agents alone and in combination with
drugs used in standard protocols, as we have previously published
and as detailed further in Core D. The end points will be growth
assays as well as biochemical and flow cytometric measurements of
apoptosis/necrosis measured at a number time points to determine
both early and late effects. Preliminary experiments have validated
the cytotoxic efficacy of some of these compounds, although their
potency appears low in some cases.
In Vivo Imaging.
[0358] The ability to simultaneously image the bio-distribution and
co-localization of cancer cells (such as ALL cells) and a
therapeutic (such as T cells or our targeted protocells), has been
hampered by the lack of multicolor luciferases with a narrow enough
emission spectra to allow spectral un-mixing using the newest
generation of optical imaging systems. As detailed in Core D, we
have developed a system to overcome this barrier, using click
beetle green (CBG) and click beetle red (CBR) luciferases that emit
in distinct parts of the spectrum with minimal spectral overlap.
Using the innovative imaging modalities in Core D, alone or
co-registered with CT, and this novel two-color biophotonic imaging
system, we will use CBG luciferase-labeled ALL cells and
quantum-dot (Qdot) or dye loaded ALL-targeted protocells to
simultaneously assess ALL disease burden, protocell trafficking,
protocell/ALL co-localization, and ultimately therapeutic efficacy
in vivo in the ALL xenografted animals. Photon intensity scales
directly with cell number and can be used to assess disease burden
and therapeutic response. Our current focus is on CRLF2 and we are
modeling the bio-distribution and co-localization of non-targeted
and CRLF2-targeted protocells labeled with Qdots or fluorescent
dyes (FIG. 22). In our first in vivo experiments, we encapsidated a
far-red fluorescent dye (AlexaFluor 680) in non-targeted protocells
and were in fact able to distinguish a red protocell signal
distinct from the CBG+ ALL cells (FIG. 22). As we continue these
studies with ALL-targeted protocells, we will utilize Qdot
technology, as Qdots are very bright, have excellent tissue
penetration, and very sharp emission peaks, making them ideal as
the second color in our biophotonic in vivo imaging system. Our
preliminary experience suggests a Qdot 705 would be ideal,
depending on size and protocell loading considerations. We will
initially test ALL lines (as detailed in FIG. 23) and then validate
our findings using CBG+ primary human ALL xenografts. We will look
for ALL/ALL-targeted protocell co-localization and correlate those
findings with therapeutic efficacy (Aim 2c). Iterative interactions
between Aim 1 and Aim 2 will allow us to determine how protocell
modification influences nanoparticle trafficking in vivo, allowing
us to optimize the final protocell design (see Table 1, Aim 2b), as
needed, to improve trafficking and co-localization with ALL. In
addition to the 18 new primary human high-risk ALL xenograft models
that we have created with a spectrum of CRLF2/JAK mutations, we
have prepared new CBG/GFP-labeled human ALL cell lines and we have
established ALL xenografts for in vivo studies from these lines
(FIG. 23).
In Vivo Toxicology.
[0359] For toxicology studies there are two major issues which must
be addressed: silica loading in the tissues, derived from the
mesoporous silica core of the protocell, and non-targeted or "off
target" delivery of the therapeutic cargos to normal tissues. In
each case, our preliminary data suggest that the liver will be the
principal target. Nevertheless, we will systematically evaluate all
major organs and tissues in treated mice for silica deposition and
cellular damage after treatment with loaded and unloaded
protocells. There are multiple lines of evidence that protocells
will have low and acceptable toxicity profiles in vivo: 1) silica
is accepted as "Generally Recognized As Safe" (GRAS) by the US Food
and Drug Administration (FDA); 2) recently, solid, dye-doped silica
nanoparticles have received approval from the FDA for targeted
molecular imaging of cancer; 3) compared to solid silica
nanoparticles, mesoporous silica nanoparticles exhibit reduced
toxicity/hemolytic activity due their surface porosity lowering the
contact area of surface silanols with cell membranes; 4) in the
case of protocells, the supported lipid bilayer reduces
silica/membrane interactions and confers safety profiles and
immunological behavior comparable to liposomes (FIG. 24); and 5)
the high internal surface area (>1000 m.sup.2/g and
ultra-thinness of the silica walls (<2-nm) of the porous silica
core result in a high dissolution rate and soluble silica, e.g.
Si(OH).sub.4, has extremely low toxicity. As a benchmark, we
compared oxidative stress and cell viability of neutral, positively
and negatively-charged targeted protocells with corresponding
liposomes and other control particles at 10.sup.9 particles/mL
corresponding to .about.1-2 .mu.g/mL (FIG. 24). The targeted,
zwitterionic DOPC protocells, which are the focus of our proposed
studies, show minimal effects on viability and reactive oxygen
species (ROS) generation. In preliminary studies (FIG. 25), we also
compared the IgG response of C57Bl/6 mice immunized twice with 10
.mu.g doses of targeted and SP94-targeted protocells to that of
virus-like particles (VLPs) conjugated to the same concentration of
targeting peptide (MS2 SP94). Targeting peptides conjugated to
lipid bilayers elicit only a weak response, because they do not
support T cell help needed for higher affinity IgG, whereas
targeting ligands displayed on VLPs induce a strong IgG response.
When considering therapeutically administered doses, for the
hepatocellular carcinoma cell line Hep3B, the LC50 and LC90 values
of free DOX are 150 and 500 ng/mL, respectively. As we have
demonstrated, when using targeted protocells, these values fall to
6 ng/mL and 20 ng/mL due to the protocell capacity, stability, and
internalization efficiency. If only a few percent of protocells are
delivered to the ALL target, then the needed administered doses of
protocells are less than about 100 .mu.g/ml, where numerous studies
have shown insignificant toxicity. To assess organ damage after
novel agent treatment, organs from treated and control mice will be
harvested and fresh frozen or fixed in formalin for histopathology.
As discussed above, Si loading will be assessed by ICP-MS. If
abnormalities are identified, we will use tissue specific stains
and electron microscopy to determine the underlying
pathophysiologic effects of the protocells. We will compare the
toxicity of targeted and non-targeted protocells and compare
protocells with encapsidated DOX with intravenous DOX provided as
free drug at an equivalent dose (typically 0.2 mg/mouse). We will
also assess if there is different toxicity in mice xenografted with
hepatotropic Nalm-6) and non-hepatotropic (380) ALL cell lines. As
the liver is the principal nonspecific target organ of the
protocells, we will assess hepatocellular damage with serum
transaminases (ALT, AST), cholestasis with bilirubin, and liver
function with prothrombin time testing.sup.50 and albumin before
and 1-7 days after exposure to nanoparticles at single and weekly
doses. In addition, we will assess the hematologic toxicity of
DOX-loaded and unloaded protocells. Based on our prior studies
evaluating the toxicity of different nanoparticle delivery systems,
20 mice will be needed for multi-compartment toxicity analysis in
each condition with each mouse strain (see Vertebrate Animals and
Core D). We will expand this number if needed. We will not only
assess toxicity in the NSG xenografts, but also in other strains
including BALB/-C, Emu-ret, Rag1ko, and MRL-lpr, allowing us to
determine if the toxicity of protocells is different in immune
competent, immune deficient, and/or in mice with activated immune
systems due to autoimmune disease.
Example 6
Combinations of Peptides can be Used to Direct Targeting and
Internalization for Non-Internalized Receptors
[0360] As shown in FIG. 26, peptides displayed on a fluid surface
are able to retain high affinity binding with low peptide densities
due to recruitment and multivalency effects By locally
concentrating peptides on protocell surface while maintaining
fluidity, differential binding affinities to target cells can be
increased over 105 relative to non-target cells. However, many
targeting peptides may not trigger internalization. The solution to
this problem is to utilize an additional peptide to promote
internalization. FIG. 26 shows Internalization efficacy of
CRLF-2-targeted protocells in the presence of the R8 peptide.
Example 7
Biophotonic Imaging in Murine ALL Xenograft Model
[0361] Protocells comprised of fluorescent label and made in
accordance with the technique described below were tested in a
murine luminescent leukemia model, as illustrated in FIGS.
27-29.
[0362] Our discovery of these novel ALL subtypes, together with our
preliminary studies demonstrating a lack of efficacy of JAK kinase
inhibitors as single agents in our xenograft models of human ALL
containing CRLF2 and JAK mutations, and the observation that a
large percentage of high risk B-precursor ALL samples express
measurable levels of CRLF2 mRNA compared to normal B cells and
respond to TSLP, leads us to hypothesize that CRLF2 is a superior
target for therapy in high-risk ALL. In order to expand the
universe of potential molecular targets with a parallel increase in
leukemic subtypes that are amenable to treatment, as well as to
allow for simultaneous targeting with multiple classes of particles
we propose to also build protocells engineered to target molecules
expressed on a wider class of ALL blasts and B cell malignancies,
including CD19 and CD22.
[0363] Included in these are primary human ALL samples from
patients in the "kinase-active" group of ultra-high risk ALL
patient. Collaborating with COG and the NCI TARGET initiative, we
received 21 ALL samples in each of 4 combinations of CRLF2 high/low
and JAK2 mutant/normal. Eighteen of these 21 samples were
established as re-transplantable ALL xenografts in highly
immunodeficient NSG (NOD-SCID/gamma common knockout) mice. We
successfully created xenografts from each of the 4 subtypes,
allowing expansion of these cells for in vitro and in vivo studies.
This is the approach we have used successfully in testing signal
transduction inhibitors, including mTOR inhibitors, as well as
engineered anti-ALL (CD 19-directed) T cell therapy, as ALL
therapeutics. 13
[0364] These have been studies with strong mechanistic analysis but
also with a clear translational focus. The second area of
innovation concerns the development of novel in vivo imaging
approaches in xenograft models. Firefly luciferase is widely and
successfully used to detect cells using biophotonic imaging in the
live animal and is a powerful approach to assess disease burden and
to image anatomic localization of labeled cells. In systems where
the experimental question is colocalization of cancer cells and a
therapeutic such as T cells or nanoparticles, an approach which
allows simultaneous detection of two different cell or particle
types would be an important methodological advance. This has been
hampered by the lack of multicolor luciferases with a narrow enough
emission spectra to allow spectral unmixing using the newest
generation of optical imaging systems (spectral unmixing is
conceptually similar to compensation in flow cytometry). We have
recently developed a system to address this, using click beetle
green and click beetle red luciferases (CBG and CBR) that emit in
distinct colors with minimal spectral overlap. An example of the
power of this approach is shown in FIGS. 27-29, where engrafted
human ALL cells and human T cells expressing CBG and CBR are both
separately and simultaneously imaged in the living mouse. Photon
intensity scales directly with cell number and can be used to
assess disease burden and response (FIGS. 27-29).
Example 8
Virus-Like Particles (VLPs) of Bacteriophage MS2 for Selection of
Peptides that Bind to ALL-Specific Targets
[0365] We have generated random peptide libraries displayed on VLPs
of the icosahedral RNA bacteriophage MS2. Caldeira J, Peabody D.
2007. Stability and assembly in vitro of bacteriophage PP7
virus-like particles. Journal of Nanobiotechnology 5: 10 Peabody D
S, Manifold-Wheeler B, Medford A, Jordan S K, do Carmo Caldeira J,
Chackerian B. 2008. Immunogenic display of diverse peptides on
virus-like particles of RNA phage MS2. Journal of Molecular Biology
380: 252-63). In general, phage display depends on (i) the ability
to genetically fuse peptides to a viral structural protein so that
they are presented in an accessible form on the surface of the
viral particle and (ii) the specific encapsidation of the nucleic
acid that encodes the peptide-protein fusion, which provides a
means to amplify affinity-selected sequences. Here we briefly
describe how we have genetically engineered the MS2 VLP to display
diverse peptides and encapsidate the mRNAs that encode them before
describing proposed affinity selection experiments.
[0366] The MS2 capsid is composed of 90 coat protein dimers that,
when expressed from a plasmid in E. coli, spontaneously
self-assemble into an icosahedral shell that is 27.5-nm in
diameter. Since the wild-type dimer does not generally tolerate
peptide insertions, we genetically fused the C-terminus of the
upstream monomer to the N-terminus of the downstream monomer so
that both halves of the dimer are produced as a single polypeptide.
We have found that the resulting `single-chain dimer` (sc-dimer),
tolerates >90% of randomized 6-mer, 8-mer, and 10-mer inserts
and yields properly assembled VLPs, each of which displays 90
copies of a foreign peptide on its surface. Id. We have also
demonstrated that each VLP encapsidates its own mRNA. Id. This
ensures that the nucleotide sequence that encodes a recombinant VLP
is contained within the particle itself and can be recovered by
reverse transcription and polymerase chain reaction (RT-PCR),
making possible the affinity selection scheme illustrated in FIG.
30.
[0367] In the `VLP display` process, random sequence libraries are
subjected to selection against the target molecule or cell, and
amplification and re-cloning of the selected sequences leads to
identification of peptide ligands specific for the target.
Chackerian B, Caldeira Jd C, Peabody J, Peabody D S. 2011. Peptide
Epitope Identification By Affinity-Selection On Bacteriophage MS2
Virus-like Particles. Journal of Molecular Biology In Press,
Accepted Manuscript; Carnes E C, Lopez D M, Donegan N P, Cheung A,
Gresham H, Timmins G S, Brinker C J. 2010. Confinement-induced
quorum sensing of individual Staphylococcus aureus bacteria. Nat
Chem Biol 6: 41-5.
[0368] To facilitate library construction and screening, we
constructed a plasmid (pDSP62) that expresses high levels of MS2
coat protein from the bacteriophage T7 promoter. This vector
normally replicates using a ColE1 origin but additionally contains
a M13 origin so that a single-stranded version of the plasmid can
be produced after super-infection with a M13 helper phage. This
enables production of complex random sequence libraries via in
vitro extension of mutagenic primers on circular single-stranded
templates using an efficient mutagenesis procedure. To restrict
insertion of peptides to the AB-loop of the downstream half of the
sc-dimer, the upstream copy is a synthetic `codon-juggled` coat
sequence containing the maximum possible number of silent
mutations. Thus, mutagenic primers can be targeted to anneal
specifically to the downstream site. Using this vector, random
sequence 6-mer, 7-mer, 8-mer, and 10-mer libraries containing
>10.sup.10 individual members have been produced. (6) MS2 VLPs
normally display 90 peptides per particle, making it difficult to
discriminate peptides with high intrinsic binding affinities from
those that have low affinity but bind with high avidity by virtue
of multiple weak interactions.
[0369] To introduce valency control into the MS2 system, we
constructed a second vector (pDSP62(am)) that encodes an alternate
version of the sc-dimer with a stop codon separating its two
halves. This mutant normally produces only wild-type coat protein
from its upstream half, but, in the presence of a non-sense
suppressor tRNA, a small percentage of ribosomes read-through the
stop codon to produce the entire sc-dimer with its guest peptide
(the peptide is displayed only in the downstream half). Both the
wild-type and sc-dimer proteins are synthesized from a single mRNA,
which they encapsidate when they co-assemble into a mosaic VLP that
displays, on average, 3 peptides per particle. Using the MS2 VLP
system, three or four rounds of affinity selection against
antibodies with known epitopes (e.g. the anti-FLAG antibody, M2)
yield peptides that closely mimic those epitopes. Chackerian B,
Caldeira Jd C, Peabody J, Peabody D S. 2011. MS2 VLP random
sequence libraries are subjected to affinity selection to identify
peptides that target surface receptors expressed specifically by
leukemia cells.
[0370] To accomplish this, we have cloned CRLF2 into a
retroviral-based expression system, infected cultured cells that
lack endogenous expression (BaF3, a murine IL3-dependent pro-B cell
line), and selected stable transfectants. Cells infected with the
CRLF2 virus express high levels of the protein, which is accessible
to extracellular antibodies and is, therefore, properly trafficked
to the membrane. Rather than performing differential selections
against malignant and normal cells (which we have found results in
a large number of peptides that bind to unsuitable targets), we
will employ BaF3-CRLF2 cells in positive selections and parental
BaF3 cells in counter-selections. We have, additionally, fused the
extracellular domain of CRLF2 to GST, which will allow for
bead-based selection strategies.
Development of B Cell-Specific Targeting Antibody Fragments.
[0371] Single-chain antibody (scFv) display is generally
accomplished by genetically fusing the foreign sequence to the
C-terminus of a phage coat protein. However, in the case of MS2
VLPs, the presence of a scFv fusion on every copy of coat protein
will likely interfere with capsid assembly. Therefore, in this aim,
we will attempt to produce VLPs that display scFvs by inserting a
stop codon in between the foreign protein and the C-terminus of
coat protein. As described above, addition of a
nonsense-suppressing tRNA will cause occasional read-through of the
stop codon and production of the fusion protein. Suppression is
relatively inefficient, however, so only a few percent of coat
protein molecules will contain the C-terminal extension.
Co-assembly of wild-type and fusion proteins should produce VLPs
with an average of 3-6 scFvs per particle. If this strategy is
successful, we will generate a randomized scFv library using the
vectors described above and perform affinity selections against CHO
cells transfected to express the B cell-specific surface antigen,
CD19.
Example 9
Development of Targeting Ligands
[0372] Preliminary proof-of-principle approaches were designed to
design and synthesize particles targeting CRLF2, an antigen
expressed on a subset of very-high risk pediatric ALLs with a very
poor outcome. To identify CRLF2-specific targeting peptides, we
have used both commercial M13 peptide libraries and we have
developed a novel method--bacteriophage MS2 virus-like peptide
(VLP) displays (detailed in FIG. 31)--to screen for peptides
against Ba-F3 cells engineered to stably express human CRLF2.
Peptides selected by affinity for Ba-F3-CRLF2 cells were
counter-selected against parental Ba-F3 cells to eliminate any
phage binding receptors common to both cell types, creating an
ideal model for counter-selections. We find that a matched
selection/counter-selection pair at very high stringency greatly
increases the specificity of the affinity selection process.
[0373] It is important to note that in this novel method, each VLP
encapsidates its own mRNA (FIG. 31) such that the nucleotide
sequences encoding any particular VLP as well as the targeting
peptide or protein are contained within the particle itself, and
can be recovered by reverse transcription and polymerase chain
reaction. Amplification and re-cloning of the selected sequences
leads to the identification of peptide ligands specific for the
target. Using a locally engineered modified pDSP62 system, random
sequence 6mer, 7mer, 8mer and 10mer libraries containing more than
10.sup.10 individual members have been produced. The high density
of MS2 VLP display (90 peptides per particle) can make it difficult
during affinity selection to discriminate peptides with high
intrinsic binding affinities from those that have low affinity, but
bind with high avidity by virtue of multiple weak interactions, but
we have overcome this problem by introducing a valency control in
the MS2 system, by making an alternate version of the single
chain-dimer with a stop codon separating its two halves; this
mutant normally produces only wild-type coat protein from its
upstream half, but in the presence of a nonsense suppressor tRNA, a
small percentage of ribosomes read through the stop codon to
produce the entire single chain-dimer with its guest peptide. Both
the wild-type and single chain-dimer proteins are synthesized from
a single mRNA, which they encapsidate when they co-assemble into a
mosaic VLP that displays about three peptides per particle on
average. Through this approach, 12 potential CLRF2 targeting
peptides were identified. Their specificity for CRLF2 has been
further demonstrated by their ability to bind the purified protein
in vitro (data not shown). Affinity selections conducted in our
laboratories have identified a peptide ligand to CRLF2 (TDAHASV)
(FIG. 31; demonstrating a Kd of 27.9 nM with no showing significant
binding to the BaF3 parental line (Kd of <3 .mu.M). As detailed
below, this CRLF2 targeting peptide has already been conjugated to
protocells and we have demonstrated selective binding and toxicity
in CRLF2-expressing ALL cell lines. We have engineered retroviruses
that direct the expression of both CD19 and CD22, and will
construct T cell lines that express high levels of ectopic protein
on their surface. These cells will be used to identify and
characterize peptide targeting ligands in a manner identical to
what we have demonstrated for CRLF2.
Single Chain Antibody Fragments (CD19, CD22).
[0374] Monoclonal antibodies directed towards B cell-specific cell
surface antigens represent an additional source of targeting agents
that can be exploited for nanotherapeutic approaches against a
broader range of B cell malignances. We have already developed a
similar strategy against CD19 to develop therapeutic T cells.
Compared to peptides, antibodies offer the prospect of
high-affinity binding even when presented at low valency on
nanoparticles, and, in many instances detailed knowledge of binding
targets and internalization properties are known.
[0375] We recently adapted the MS2 VLP.sup.6,7 for display of
antibody fragments by fusing the coding sequence for a single-chain
antibody fragment (scFv) to the MS2 coat protein. The particles as
designed to display a controlled number of scFv's per particle. We
have so far fused several different scFv's to coat protein and
demonstrated the ability of the VLP-scFv to bind its target. Based
on published amino acid sequences, we synthesized (using assembly
PCR) an E. coli codon-optimized DNA sequence that encodes the
anti-CD 19 protein and fused it to the C-terminus of the MS2 coat
protein sequence with an amber codon at the fusion junction. When
this gene is expressed in bacteria with a suppressor tRNA, it
produces large amounts of single-chain coat protein, and small
amounts (a few percent) of the coat-scFv fusion, which co-assemble
to yield a VLP displaying a few antibodies per particle, on
average. FACS analysis shows that the CD19-specific scFv directs
VLPs to bind CD19+NALM6 B-ALL cells (FIG. 32), but not to cells
lacking CD 19 expression. Future studies will characterize the
affinity of the interaction and more carefully document its
specificity. A similar VLP displaying anti-CD22 has been
constructed, and, as with the anti-CD19 particle, will be
characterized both for the affinity and the specificity of its
interaction with cell lines specifically expressing these antigens.
These scFv constructs will then be conjugated to protocells.
Aim 1b. Targeted Protocell Production and Optimization.
[0376] Protocell nanoporous silica cores will be synthesized by
self-assembly and loaded with cargos by immersion; their supported
lipid bilayers will be modified with targeting and fusogenic
peptides, single-chain antibodies, and PEG to create sets of
targeted nanoparticles. Further optimization will be accomplished
by determining cargo content, determining the necessary extent of
modification with fusogenic peptides and poly(ethylene glycol)
(PEG), pore size, and solubility of the nanoporous silica core
(which controls loading and release rates). Protocells will be
studied in vitro in ALL cell lines using flow cytometry and
hyperspectral fluorescence confocal microscopy.
Protocell Binding, Specificity, Internalization and
Cytotoxicity:
[0377] Protocells are synthesized via fusion of liposomes to
spherical, nanoporous silica cores (100-150 nm in diameter) that
are pre-loaded via simple immersion in a solution of the desired
cargos. Based on optimization studies aimed to maximize colloidal
stability and cargo retention in simulated body fluids and minimize
non-specific interactions with serum proteins and non-targeted
cells, we utilized the following supported lipid bilayer (SLB)
composition in all surface-binding, internalization, and cargo
delivery experiments: DOPC (T.sub.m=-20.degree. C.) or DPPC
(T.sub.m=44.degree. C.) with 5 wt % DOPE 30 wt % cholesterol, and 5
wt % 18:1 (or 16:0) PEG-2000 PE. Using a hetero-bifunctional
crosslinker with a PEG (n=24) spacer, SP94 peptides (a targeting
ligand specific for hepatocellular carcinoma cells (HCC) identified
via filamentous phage display detailed in were covalently
conjugated to DOPE moieties in the SLB at concentrations ranging
from 0.002-5.0 wt % (corresponding to 1-2048 peptides per particle,
on average). 120-nm liposomes with identical bilayer compositions
were synthesized for comparative purposes. FIG. 33A depicts the
successive stages of protocell binding (step 1), internalization
(step 2), endosomal escape (step 3), and nuclear targeting of
desired cargo(s) (step 4) by which protocells selectively deliver
encapsulated cargos to a cell of interest. Importantly, the fluid
but stable SLB enables targeting peptides to be recruited to cell
surface receptors. This promotes high avidity multivalent binding
and internalization by receptor mediated endocytosis. Dissociation
constants (Kd, where Kd is inversely related to affinity) were used
to quantify surface binding of SP94-targeted protocells to
hepatocellular carcinoma cells (Hep3B), normal hepatocytes,
endothelial cells, and immune cells..sup.1 Protocells modified with
only six SP94 peptides per particle exhibit a 10,000-fold greater
affinity for Hep3B than for normal hepatocytes, and other control
cells (FIG. 34a), providing the specificity necessary for
efficacious targeted delivery..sup.1 Furthermore, SP94-modified
protocells have a 200-fold higher affinity for Hep3B than free
SP94, a 1000-fold higher affinity for Hep3B than nanoparticles
bearing of a non-targeting control peptide, and a 10.sup.4 higher
affinity for Hep3B than unmodified particles (FIG. 34a).
[0378] Importantly, the affinity of protocells is a function of
both peptide density and the fluidity of the supported lipid
bilayer, and the dissociation constant (K.sub.d) can be precisely
controlled by changing the composition of the bilayer to include
varying amounts of fluid and non-fluid lipid components. To
demonstrate that binding results in internalization and cytosolic
delivery of multiple cargos, FIG. 34b shows hyperspectral confocal
images of four categories of fluorescently labelled cargo mimics
delivered by a single targeted protocell. After 4 hours, calcein (a
drug mimic), ds-DNA (an siRNA mimic), red fluorescent protein (a
toxin mimic), and quantum dots are delivered into the cytosol.
Calcein and dsDNA (both conjugated with a nuclear localization
sequence) are further delivered into the nucleus. Delivery profiles
are controlled by the pore size and solubility of the silica core
along with protocell surface modification with a fusogenic peptide
that promotes osmotic swelling and endosomal disruption (see FIG.
33A, step 3).
[0379] FIG. 34b compares the percentage of viable multi-drug
resistant Hep3B or hepatocytes after exposure to LC.sub.90 or
LC.sub.50 concentrations of the chemotherapeutic drug doxorubicin
(or a drug cocktail) delivered in targeted DOPC (fluid) protocells,
DOPC liposomes, or state of the art DSPC (non-fluid) liposomes. For
DOPC protocells+DOX, we observe spectacular MDR1.sup.+
Hep3B-specific cytotoxicity, whereas for corresponding DOPC
liposomes+DOX the results are comparable to free DOX. This
difference is attributable in part to fluid liposomes being
unstable and leaking their cargo. Even stable DSPC liposomes,
however, show substantially inferior properties. These data reveal
that the combined capacity, stability, and targeting efficacy of
the protocell allow it to significantly outperform liposomal
delivery agents. In fact a single protocell loaded with a drug
cocktail is able to kill a drug-resistant HCC cell, representing a
million-fold improvement over comparable liposomes
CRLF2-Targeted Protocells.
[0380] The CRLF2-targeting peptide shown in FIG. 31 was conjugated
to protocells. CRLF2-targeted protocells were demonstrated to
possess a 1,000-fold higher affinity for engineered BaF3-CRLF2
cells expressing high levels of CRLF2 (FIG. 35), and for the MUTZ5
or MHHCALL4 cells (established human ALL cells lines with high
CRLF2 expression and JAK kinase mutations) (FIG. 36), when compared
to untargeted protocells, the parental BAF3 cell line, or the
CRLF2-negative NALM6 ALL cell line which served as controls (FIGS.
35 and 36). This affinity was also achievable at very low peptide
densities due to the fluid protocell surface, potentially
minimizing non-specific binding and/or immune responses. Targeted
protocells loaded with doxorubicin (which is intrinsically
fluorescent) were able to selectively bind to cells expressing
CRLF2, and after incubation at 37.degree. C., to become
internalized and deliver drug to the cytoplasm of the cells within
24 hours while showing no non-specific interactions with control
cells (FIG. 35B, FIG. 36). Further, modification of the protocell
surface with an octa-arginine (R8) peptide promoted this selective
internalization in a density-dependent manner, proving that
protocells support complex synergistic interactions enabling
targeting and internalization for cancers whose targeting peptides
might poorly internalized (FIG. 35C).
[0381] We are excited that these preliminary studies demonstrate
that we can selectively target CRLF2-expressing ALL cells with
CRLF2-targeted nanocarriers, that the protocell and its drug cargo
is internalized, and taken up by the cytoplasm. An additional
series of studies will investigate the added benefit of conjugating
multiple targeting peptides to the surface of a single particle. We
postulate that protocells that recognize both CD 19 and CD22 might
have an improved therapeutic index compared to those that target a
single antigen, and might limit the potential emergence of
resistant lines that can arise by loss of a single surface
protein.
Defining Optimal Chemotherapeutic Cargoes and Determining
Therapeutic Efficacy of ALL-Targeted Protocells In Vitro.
[0382] The ability of protocells to protect their therapeutic cargo
until released within the target cell and to deliver multiple
cargoes are being exploited to determine the most efficacious drug
combinations for packaging into ALL-targeted protocells. Using the
in vitro ALL cell line models described above in Aim 1b), we are
testing traditional ALL therapeutic drug combinations (FIG. 37) as
well as novel compounds that demonstrate efficacy against resistant
forms of high-risk ALL (such as the mTOR pathway inhibitor
sirolimus) that we have identified in high throughput screens or
ALL xenograft models (FIG. 38). The therapeutic efficacy of drugs
encapsidated in protocells is being compared to exposure of the
cell lines to free drug(s) using cell biologic, flow cytometric,
and phosphoflow cytometric assays, allowing us to test and model
pharmacodynamic assessments of target inhibition in ALL cells in
vitro.
[0383] When CRLF2-targeted protocells with encapsidated doxorubicin
were incubated with the CRLF2-expressing cell line MHHCALL4,
binding, protocell and drug uptake, and doxorubicin release into
the cytoplasm could be demonstrated in CRLF2-expressing cells but
not in control cells. Although CRLF2-expressing high-risk ALL
patients tend to be resistant to intensive therapeutic
regimens,.sup.9-11 we demonstrated in very preliminary studies that
after uptake and drug delivery, CRLF2-targeted protocells with
encapsidated doxorubicin promoted rapid apoptosis and cell death in
MHH CALL4 cells (FIG. 37) In order to validate compounds and
ALL-targeted/drug-loaded protocell designs as being cytotoxic to
ALL cells, and to choose those compounds which are most active in
vitro (Aim 1c) for further screening in vivo in xenograft models
(Aim 2), it is necessary to assess response and targeting behavior
in human ALL cell lines. This is an approach that our team of
investigators have used successfully to prioritize compounds for
our ALL xenograft experiments, leading to our current trials of
mTOR inhibitors in ALL. In Aim 1c), we will validate the cytotoxic
activity of mTOR inhibitors in high-risk ALL cases (using cell
lines reflective of the CRLF2/JAK genotype/phenotype), based on
preliminary xenograft data that demonstrate activity of this class
of drugs in this subset of leukemias. All lines used will include
CD19+ and CD19- or CD22+ and CD22- variants of T cell lines, and
CRLF2+ and CRLF2- variants of the same line (NALM6 is
CRLF2-negative and we have engineered a CRLF2+NALM-6 line marked
with GFP and Click Beetle Green luciferase figures s3 and s4,
below)).
[0384] Our panel will also include the high CRLF2-expressing/JAK
mutated MUTZ5 and MHHCALL4 human ALL cell lines which are markedly
drug resistant and that express both CD19 and CD22. In addition a
variety of ALL cell lines that express CD19 and/or CD22 will be
used in cytotoxicity experiments. The development of reagents that
recognize antigens present on a very large proportion of B-cell
leukemias and lymphomas will dramatically increase the scope of our
previous work that targeted a very narrow cohort of pediatric ALLs
expressing CRLF2. These studies will include comprehensive
dose-response curves using the agents alone and in combination with
drugs used in standard protocols, as we have previously published.
The end points will be growth assays as well as biochemical and
flow cytometric measurements of apoptosis/necrosis measured at a
number time points to determine both early and late effects.
Preliminary experiments have validated the cytotoxic efficacy of
some of these compounds, although their potency appears low in some
cases. If this continues to be the case, we can make a compelling
argument for protocell based delivery that results in elevated
intracellular concentrations compared to what can be achieved
following systemic administration. Each of the protocell variants
developed in Aims 1a) and 1b) will be tested: free compound,
compound loaded into protocells, and targeted nanocarriers against
CD 19 and CD22 using either single chain antibodies or targeting
peptides. If experiments in Aim 1a) and 1b) show that
internalization is improved using an anti-CD22 targeting agent (as
CD22 is known to be rapidly internalized after antibody binding),
then we will test that construct as well. The goal of Aim 1c) is to
find the most cytotoxic combinations of targeting ligand, cargo,
and protocell to test in vivo in ALL xenograft models.
Example 10
Model CRLF2 and CD99+ Cell Lines for Selection and In Vitro and In
Vivo Studies
[0385] Fluorescently tagged NALM6 cells were transduced with a
retrovirus directing the expression of ectopic human CRLF2 and
stable clones with a 10-fold increase in surface expression (FIG.
39A) were established. This increase in expression is adequate for
initial experiments and these cells have been used in preliminary
studies using chick embryos. In addition, these cells will be
evaluated for their ability to form xenografts with characteristics
similar to those of the parent cells. We also constructed similar
viral constructs encoding human CD99 as proposed in the last
progress report and established a series of fluorescent NALM6
clones that with very high levels of ectopic gene expression.
However, we failed to detect trafficking of CD99 to the cell
surface based on a flow analysis. An alternative cloning strategy
to allow for proper CD99 trafficking and increased relative levels
of surface CRLF2 in the cell lines that will be used in the in vivo
assays is now being used.
[0386] A sequence listing of all sequences disclosed in the present
application follows:
TABLE-US-00001 Sequence Listings of Peptides/Nucleotides: SEQ ID
NO: 1 MTAAPVHGGHHHHHH SEQ ID NO: 2 RRRRRRRRGGC SEQ ID NO: 3
GLFHAIAHFIHGGWHGLIHGWYGGGC SEQ ID NO: 4 MTAAPVH SEQ ID NO: 5
LTTPNWV SEQ ID NO: 6 AAQTSTP SEQ ID NO: 7 TDAHASV SEQ ID NO: 8
FSYLPSH SEQ ID NO: 9 YTTQSWQ SEQ ID NO: 10 MHAPPFY SEQ ID NO: 11
AATLFPL SEQ ID NO: 12 LTSRPTL SEQ ID NO: 13 ETKAWWL SEQ ID NO: 14
HWGMWSY SEQ ID NO: 15 SQIFGNK SEQ ID NO: 16 SQAFVLV SEQ ID NO: 17
WPTRPWH SEQ ID NO: 18 WVHPPKV SEQ ID NO: 19 TMCIYCT SEQ ID NO: 20
ASRIVTS SEQ ID NO: 21 WTGSYRW SEQ ID NO: 22 NILSLSM SEQ ID NO: 23
RRRRRRRR SEQ ID NO: 24 GLFHAIAHFIHGGWHGLIHGWY SEQ ID NO: 25
WPTXPW[--H] SEQ ID NO: 26 ---S[FW][ST]XWXX--WX------ SEQ ID NO: 27
-----XSPXXWXXXXX------- SEQ ID NO: 28
GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY SEQ ID NO: 29 RRMKWKK SEQ
ID NO: 30 PKKKRKV SEQ ID NO: 31 KR[PAATKKAGQA]KKKK SEQ ID NO: 32
acatgaggat tacccatgt SEQ ID NO: 33 acatgaggat cacccatgt SEQ ID NO:
34 FS--YLP[--S][--H] SEQ ID NO: 35 MT-AAP[VFW]H
* * * * *
References